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Sample records for aureus biofilm maturation

  1. Staphylococcus aureus biofilms

    PubMed Central

    Archer, Nathan K; Mazaitis, Mark J; Costerton, J William; Leid, Jeff G; Powers, Mary Elizabeth

    2011-01-01

    Increasing attention has been focused on understanding bacterial biofilms and this growth modality's relation to human disease. In this review we explore the genetic regulation and molecular components involved in biofilm formation and maturation in the context of the Gram-positive cocci, Staphylococcus aureus. In addition, we discuss diseases and host immune responses, along with current therapies associated with S. aureus biofilm infections and prevention strategies. PMID:21921685

  2. Hyaluronan Modulation Impacts Staphylococcus aureus Biofilm Infection.

    PubMed

    Ibberson, Carolyn B; Parlet, Corey P; Kwiecinski, Jakub; Crosby, Heidi A; Meyerholz, David K; Horswill, Alexander R

    2016-06-01

    Staphylococcus aureus is a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in an S. aureus biofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA). S. aureus secretes HysA in order to cleave HA during infection. Through in vitro biofilm studies with HA, the hysA mutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction of hysA expression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from an hysA mutant biofilm infection compared to the S. aureus wild type. Histopathology demonstrated that infection with an hysA mutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA and S. aureus HysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of the S. aureus biofilm matrix and HysA is important for dissemination from a biofilm infection. PMID:27068096

  3. Staphopains Modulate Staphylococcus aureus Biofilm Integrity

    PubMed Central

    Mootz, Joe M.; Malone, Cheryl L.; Shaw, Lindsey N.

    2013-01-01

    Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ΔsigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture. PMID:23798534

  4. Aspartate inhibits Staphylococcus aureus biofilm formation.

    PubMed

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp. PMID:25687923

  5. Global Gene Expression in Staphylococcus aureus Biofilms

    PubMed Central

    Beenken, Karen E.; Dunman, Paul M.; McAleese, Fionnuala; Macapagal, Daphne; Murphy, Ellen; Projan, Steven J.; Blevins, Jon S.; Smeltzer, Mark S.

    2004-01-01

    We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions. PMID:15231800

  6. Statins and Antimicrobial Effects: Simvastatin as a Potential Drug against Staphylococcus aureus Biofilm

    PubMed Central

    Franco, Gilson Cesar; Schwartz-Filho, Humberto Osvaldo; de Andrade, Eduardo Dias

    2015-01-01

    Statins are important lipid-lowering agents with other pleiotropic effects. Several studies have explored a possible protective effect of statins to reduce the morbidity and mortality of many infectious diseases. Staphylococcus aureus is one of the main pathogens implicated in nosocomial infections; its ability to form biofilms makes treatment difficult. The present study observed the MIC of atorvastatin, pravastatin and simvastatin against S. aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis. Simvastatin was the only agent with activity against clinical isolates and reference strains of methicilin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Thus, the effects of simvastatin on the growth, viability and biofilm formation of S. aureus were tested. In addition, a possible synergistic effect between simvastatin and vancomycin was evaluated. Simvastatin’s MIC was 15.65 µg/mL for S. aureus 29213 and 31.25 µg/mL for the other strains of S. aureus. The effect of simvastatin was bactericidal at 4xMIC and bacteriostatic at the MIC concentration. No synergistic effect was found between simvastatin and vancomycin. However, the results obtained against S. aureus biofilms showed that, in addition to inhibiting adhesion and biofilm formation at concentrations from 1/16xMIC to 4xMIC, simvastatin was also able to act against mature biofilms, reducing cell viability and extra-polysaccharide production. In conclusion, simvastatin showed pronounced antimicrobial activity against S. aureus biofilms, reducing their formation and viability. PMID:26020797

  7. Myeloid-derived suppressor cells (MDSCs) contribute to S. aureus orthopedic biofilm infection

    PubMed Central

    Heim, Cortney E.; Vidlak, Debbie; Scherr, Tyler D.; Kozel, Jessica A.; Holzapfel, Melissa; Muirhead, David E.; Kielian, Tammy

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes that are potent inhibitors of T cell activation. A role for MDSCs in bacterial infections has only recently emerged and nothing is known about MDSC function in the context of Staphylococcus aureus (S. aureus) infection. Since S. aureus biofilms are capable of subverting immune-mediated clearance, we examined whether MDSCs could play a role in this process. CD11b+Gr-1+ MDSCs represented the main cellular infiltrate during S. aureus orthopedic biofilm infection, accounting for over 75% of the CD45+ population. Biofilm-associated MDSCs inhibited T cell proliferation and cytokine production, which correlated with a paucity of T cell infiltrates at the infection site. Analysis of FACS-purified MDSCs recovered from S. aureus biofilms revealed increased Arg-1, iNOS, and IL-10 expression, key mediators of MDSC suppressive activity. Targeted depletion of MDSCs and neutrophils using the mAb 1A8 (anti-Ly6G) improved bacterial clearance by enhancing the intrinsic pro-inflammatory attributes of infiltrating monocytes and macrophages. Furthermore, the ability of monocytes/macrophages to promote biofilm clearance in the absence of MDSC action was revealed with RB6-C85 (anti-Gr-1 or anti-Ly6G/Ly6C) administration, which resulted in significantly increased S. aureus burdens both locally and in the periphery, since effector Ly-6C monocytes and by extension, mature macrophages, were also depleted. Collectively, these results are the first to demonstrate that MDSCs are key contributors to the chronicity of S. aureus biofilm infection, as their immunosuppressive function prevents monocyte/macrophage proinflammatory activity, which facilitates biofilm persistence. PMID:24646737

  8. Synergistic activity between an antimicrobial polyacrylamide and daptomycin versus Staphylococcus aureus biofilm.

    PubMed

    Siala, Wafi; Van Bambeke, Françoise; Taresco, Vincenzo; Piozzi, Antonella; Francolini, Iolanda

    2016-07-01

    Antibiotic resistance of bacteria growing in biofilms compared to their planktonic counterparts enhances the difficulty to eradicate biofilm-associated infections. In the last decade, combination antibiotic therapy has emerged as an attractive strategy for treating biofilm infections, even if in most of tolerant biofilms the optimal combinations are still unknown. In this study, an antimicrobial cationic polyacrylamide was used in combination with daptomycin or moxifloxacin against mature biofilms of Staphylococcus aureus clinical isolates to examine a possible improvement of the antibiofilm activity of the two antibiotics. The polymer did not have an effect on moxifloxacin but significantly increased the antibiofilm efficacy of daptomycin. These findings are presumably related to the different mechanism of action of the two drugs. In summary, our data highlighted the ability of polycations to increase daptomycin antibiofilm activity providing a potential strategy to eradicate biofilms in industrial or medical settings. PMID:27154750

  9. Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases

    PubMed Central

    Traba, Christian; Liang, Jun F.

    2011-01-01

    Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: 1) killing bacteria in biofilms by causing severe cell membrane damage, and 2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum . Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections. PMID:21774615

  10. Characterization of Staphylococcus aureus Biofilm Formation in Urinary Tract Infection

    PubMed Central

    YOUSEFI, Masoud; POURMAND, Mohammad Reza; FALLAH, Fatemeh; HASHEMI, Ali; MASHHADI, Rahil; NAZARI-ALAM, Ali

    2016-01-01

    Background: The aim of this study was to investigate the antibiotic susceptibility pattern as well as the phenotypic and genotypic biofilm formation ability of Staphylococcus aureus isolates from patients with urinary tract infection (UTI). Methods: A total of 39 isolates of S. aureus were collected from patients with UTI. The antibiotic susceptibility patterns of the isolates were determined by the Kirby-Bauer disk-diffusion. We used the Modified Congo red agar (MCRA) and Microtiter plate methods to assess the ability of biofilm formation. All isolates were examined for determination of biofilm related genes, icaA, fnbA, clfA and bap using PCR method. Results: Linezolid, quinupristin/dalfopristin and chloramphenicol were the most effective agents against S. aureus isolates. Overall, 69.2% of S. aureus isolates were biofilm producers. Resistance to four antibiotics such as nitrofurantoin (71.4% vs. 28.6%, P=0.001), tetracycline (57.7% vs. 42.3%, P=0.028), erythromycin and ciprofloxacin (56% vs. 44%, P=0.017) was higher among biofilm producers than non-biofilm producers. The icaA, fnbA and clfA genes were present in all S. aureus isolates. However, bap gene was not detected in any of the isolates. Conclusion: Our findings reinforce the role of biofilm formation in resistance to antimicrobial agents. Trimethoprimsulfamethoxazole and doxycycline may be used as an effective treatment for UTI caused by biofilm producers S. aureus. Our results suggest that biofilm formation is not dependent to just icaA, fnbA, clfA and bap genes harbor in S. aureus strains. PMID:27252918

  11. Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

    PubMed Central

    Gil, Carmen; Solano, Cristina; Burgui, Saioa; Latasa, Cristina; García, Begoña; Toledo-Arana, Alejandro

    2014-01-01

    The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. PMID:24343648

  12. Involvement of Iron in Biofilm Formation by Staphylococcus aureus

    PubMed Central

    Huang, Hsiu-Yun; Cheng, Yi-Ching

    2012-01-01

    Staphylococcus aureus is a human pathogen that forms biofilm on catheters and medical implants. The authors' earlier study established that 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) inhibits biofilm formation by S. aureus by preventing the initial attachment of the cells to a solid surface and reducing the production of polysaccharide intercellular adhesin (PIA). Our cDNA microarray and MALDI-TOF mass spectrometric studies demonstrate that PGG treatment causes the expression of genes and proteins that are normally expressed under iron-limiting conditions. A chemical assay using ferrozine verifies that PGG is a strong iron chelator that depletes iron from the culture medium. This study finds that adding FeSO4 to a medium that contains PGG restores the biofilm formation and the production of PIA by S. aureus SA113. The requirement of iron for biofilm formation by S. aureus SA113 can also be verified using a semi-defined medium, BM, that contains an iron chelating agent, 2, 2′-dipyridyl (2-DP). Similar to the effect of PGG, the addition of 2-DP to BM medium inhibits biofilm formation and adding FeSO4 to BM medium that contains 2-DP restores biofilm formation. This study reveals an important mechanism of biofilm formation by S. aureus SA113. PMID:22479621

  13. Stilbenes reduce Staphylococcus aureus hemolysis, biofilm formation, and virulence.

    PubMed

    Lee, Kayeon; Lee, Jin-Hyung; Ryu, Shi Yong; Cho, Moo Hwan; Lee, Jintae

    2014-09-01

    Stilbenoids have a broad range of beneficial health effects. On the other hand, the emergence of antibiotic-resistant Staphylococcus aureus presents a worldwide problem that requires new antibiotics or nonantibiotic strategies. S. aureus produces α-hemolysin (a pore-forming cytotoxin) that has been implicated in the pathogenesis of sepsis and pneumonia. Furthermore, the biofilms formed by S. aureus constitute a mechanism of antimicrobial resistance. In this study, we investigated the hemolytic and antibiofilm activities of 10 stilbene-related compounds against S. aureus. trans-Stilbene and resveratrol at 10 μg/mL were found to markedly inhibit human blood hemolysis by S. aureus, and trans-stilbene also inhibited S. aureus biofilm formation without affecting its bacterial growth. Furthermore, trans-stilbene and resveratrol attenuated S. aureus virulence in vivo in the nematode Caenorhabditis elegans, which is normally killed by S. aureus. Transcriptional analysis showed that trans-stilbene repressed the α-hemolysin hla gene and the intercellular adhesion locus (icaA and icaD) in S. aureus, and this finding was in line with observed reductions in virulence and biofilm formation. In addition, vitisin B, a stilbenoid tetramer, at 1 μg/mL was observed to significantly inhibit human blood hemolysis by S. aureus. PMID:25007234

  14. A Combined Pharmacodynamic Quantitative and Qualitative Model Reveals the Potent Activity of Daptomycin and Delafloxacin against Staphylococcus aureus Biofilms

    PubMed Central

    Bauer, Julia; Siala, Wafi; Tulkens, Paul M.

    2013-01-01

    Biofilms are associated with persistence of Staphylococcus aureus infections and therapeutic failures. Our aim was to set up a pharmacodynamic model comparing antibiotic activities against biofilms and examining in parallel their effects on viability and biofilm mass. Biofilms of S. aureus ATCC 25923 (methicillin-sensitive S. aureus [MSSA]) or ATCC 33591 (methicillin-resistant S. aureus [MRSA]) were obtained by culture in 96-well plates for 6 h/24 h. Antibiotic activities were assessed after 24/48 h of exposure to concentrations ranging from 0.5 to 512 times the MIC. Biofilm mass and bacterial viability were quantified using crystal violet and the redox indicator resazurin. Biofilms stained with Live/Dead probes were observed by using confocal microscopy. Concentration-effect curves fitted sigmoidal regressions, with a 50% reduction toward both matrix and viability obtained at sub-MIC or low multiples of MICs against young biofilms for all antibiotics tested. Against mature biofilms, maximal efficacies and potencies were reduced, with none of the antibiotics being able to completely destroy the matrix. Delafloxacin and daptomycin were the most potent, reducing viability by more than 50% at clinically achievable concentrations against both strains, as well as reducing biofilm depth, as observed in confocal microscopy. Rifampin, tigecycline, and moxifloxacin were effective against mature MRSA biofilms, while oxacillin demonstrated activity against MSSA. Fusidic acid, vancomycin, and linezolid were less potent overall. Antibiotic activity depends on biofilm maturity and bacterial strain. The pharmacodynamic model developed allows ranking of antibiotics with respect to efficacy and potency at clinically achievable concentrations and highlights the potential utility of daptomycin and delafloxacin for the treatment of biofilm-related infections. PMID:23571532

  15. Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin

    PubMed Central

    Scherr, Tyler D.; Hanke, Mark L.; Huang, Ouwen; James, David B. A.; Horswill, Alexander R.; Bayles, Kenneth W.; Fey, Paul D.; Torres, Victor J.

    2015-01-01

    ABSTRACT The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. PMID:26307164

  16. Crystal Violet and XTT Assays on Staphylococcus aureus Biofilm Quantification.

    PubMed

    Xu, Zhenbo; Liang, Yanrui; Lin, Shiqi; Chen, Dingqiang; Li, Bing; Li, Lin; Deng, Yang

    2016-10-01

    Staphylococcus aureus (S. Aureus) is a common food-borne pathogenic microorganism. Biofilm formation remains the major obstruction for bacterial elimination. The study aims at providing a basis for determining S. aureus biofilm formation. 257 clinical samples of S. aureus isolates were identified by routine analysis and multiplex PCR detection and found to contain 227 MRSA, 16 MSSA, 11 MRCNS, and 3 MSCNS strains. Two assays for quantification of S. aureus biofilm formation, the crystal violet (CV) assay and the XTT (tetrazolium salt reduction) assay, were optimized, evaluated, and further compared. In CV assay, most isolates formed weak biofilm 74.3 %), while the rest formed moderate biofilm (23.3 %) or strong biofilm (2.3 %). However, most isolates in XTT assay showed weak metabolic activity (77.0 %), while the rest showed moderate metabolic activity (17.9 %) or high metabolic activity (5.1 %). In this study, we found a distinct strain-to-strain dissimilarity in terms of both biomass formation and metabolic activity, and it was concluded from this study that two assays were mutual complementation rather than being comparison. PMID:27324342

  17. Effects of bacteriocins on methicillin-resistant Staphylococcus aureus biofilm.

    PubMed

    Okuda, Ken-ichi; Zendo, Takeshi; Sugimoto, Shinya; Iwase, Tadayuki; Tajima, Akiko; Yamada, Satomi; Sonomoto, Kenji; Mizunoe, Yoshimitsu

    2013-11-01

    Control of biofilms formed by microbial pathogens is an important subject for medical researchers, since the development of biofilms on foreign-body surfaces often causes biofilm-associated infections in patients with indwelling medical devices. The present study examined the effects of different kinds of bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by certain bacteria, on biofilms formed by a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). The activities and modes of action of three bacteriocins with different structures (nisin A, lacticin Q, and nukacin ISK-1) were evaluated. Vancomycin, a glycopeptide antibiotic used in the treatment of MRSA infections, showed bactericidal activity against planktonic cells but not against biofilm cells. Among the tested bacteriocins, nisin A showed the highest bactericidal activity against both planktonic cells and biofilm cells. Lacticin Q also showed bactericidal activity against both planktonic cells and biofilm cells, but its activity against biofilm cells was significantly lower than that of nisin A. Nukacin ISK-1 showed bacteriostatic activity against planktonic cells and did not show bactericidal activity against biofilm cells. Mode-of-action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells. Our results suggest that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of MRSA biofilm infections. PMID:23979748

  18. Effects of Bacteriocins on Methicillin-Resistant Staphylococcus aureus Biofilm

    PubMed Central

    Zendo, Takeshi; Sugimoto, Shinya; Iwase, Tadayuki; Tajima, Akiko; Yamada, Satomi; Sonomoto, Kenji

    2013-01-01

    Control of biofilms formed by microbial pathogens is an important subject for medical researchers, since the development of biofilms on foreign-body surfaces often causes biofilm-associated infections in patients with indwelling medical devices. The present study examined the effects of different kinds of bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by certain bacteria, on biofilms formed by a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). The activities and modes of action of three bacteriocins with different structures (nisin A, lacticin Q, and nukacin ISK-1) were evaluated. Vancomycin, a glycopeptide antibiotic used in the treatment of MRSA infections, showed bactericidal activity against planktonic cells but not against biofilm cells. Among the tested bacteriocins, nisin A showed the highest bactericidal activity against both planktonic cells and biofilm cells. Lacticin Q also showed bactericidal activity against both planktonic cells and biofilm cells, but its activity against biofilm cells was significantly lower than that of nisin A. Nukacin ISK-1 showed bacteriostatic activity against planktonic cells and did not show bactericidal activity against biofilm cells. Mode-of-action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells. Our results suggest that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of MRSA biofilm infections. PMID:23979748

  19. Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm.

    PubMed

    Xu, Yuanxi; Jones, John E; Yu, Haiqing; Yu, Qingsong; Christensen, Gordon D; Chen, Meng; Sun, Hongmin

    2015-12-01

    Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections. PMID:26369955

  20. Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm

    PubMed Central

    Xu, Yuanxi; Jones, John E.; Yu, Haiqing; Yu, Qingsong; Christensen, Gordon D.

    2015-01-01

    Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections. PMID:26369955

  1. Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilms.

    PubMed

    Yang, Qingping; Phillips, Priscilla L; Sampson, Edith M; Progulske-Fox, Ann; Jin, Shouguang; Antonelli, Patrick; Schultz, Gregory S

    2013-01-01

    Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms. PMID:23927831

  2. Distribution and Inhibition of Liposomes on Staphylococcus aureus and Pseudomonas aeruginosa Biofilm

    PubMed Central

    Dong, Dong; Thomas, Nicky; Thierry, Benjamin; Vreugde, Sarah; Prestidge, Clive A.; Wormald, Peter-John

    2015-01-01

    Background Staphylococcus aureus and Pseudomonas aeruginosa are major pathogens in chronic rhinosinusitis (CRS) and their biofilms have been associated with poorer postsurgical outcomes. This study investigated the distribution and anti-biofilm effect of cationic (+) and anionic (-) phospholipid liposomes with different sizes (unilamellar and multilamellar vesicle, ULV and MLV respectively) on S. aureus and P. aeruginosa biofilms. Method Specific biofilm models for S. aureus ATCC 25923 and P. aeruginosa ATCC 15692 were established. Liposomal distribution was determined by observing SYTO9 stained biofilm exposed to DiI labeled liposomes using confocal scanning laser microscopy, followed by quantitative image analysis. The anti-biofilm efficacy study was carried out by using the alamarBlue assay to test the relative viability of biofilm treated with various liposomes for 24 hours and five minutes. Results The smaller ULVs penetrated better than larger MLVs in both S. aureus and P. aeruginosa biofilm. Except that +ULV and –ULV displayed similar distribution in S. aureus biofilm, the cationic liposomes adhered better than their anionic counterparts. Biofilm growth was inhibited at 24-hour and five-minute exposure time, although the decrease of viability for P. aeruginosa biofilm after liposomal treatment did not reach statistical significance. Conclusion The distribution and anti-biofilm effects of cationic and anionic liposomes of different sizes differed in S. aureus and P. aeruginosa biofilms. Reducing the liposome size and formulating liposomes as positively charged enhanced the penetration and inhibition of S. aureus and P. aeruginosa biofilms. PMID:26125555

  3. Alpha-toxin promotes Staphylococcus aureus mucosal biofilm formation.

    PubMed

    Anderson, Michele J; Lin, Ying-Chi; Gillman, Aaron N; Parks, Patrick J; Schlievert, Patrick M; Peterson, Marnie L

    2012-01-01

    Staphylococcus aureus causes many diseases in humans, ranging from mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may be asymptomatically carried in the anterior nares or vagina or on the skin, serving as a reservoir for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and the leading cause of TSS. The cytolysin α-toxin (also known as α-hemolysin or Hla) is the major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. The current study aims to characterize the differences between TSS USA200 strains [high (hla(+)) and low (hla(-)) α-toxin producers] in their ability to disrupt vaginal mucosal tissue and to characterize the subsequent infection. Tissue viability post-infection and biofilm formation of TSS USA200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hla(-)), MNPE (hla(+)), and MNPE isogenic hla knockout (hlaKO), were observed via LIVE/DEAD® staining and confocal microscopy. All TSS strains grew to similar bacterial densities (1-5 × 10(8) CFU) on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587 (hla(-)), MN8 (hla(-)), nor MNPE hlaKO formed biofilms. The latter strains were also less cytotoxic than wild-type MNPE. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. We speculate that α-toxin affects S. aureus phenotypic growth on vaginal mucosa by promoting tissue disruption and biofilm formation. Further, α-toxin mutants (hla(-)) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV). PMID:22919655

  4. Antimicrobial activity of essential oils against Staphylococcus aureus biofilms.

    PubMed

    Vázquez-Sánchez, Daniel; Cabo, Marta L; Rodríguez-Herrera, Juan J

    2015-12-01

    The present study was aimed to evaluate the potential of essential oils to remove the foodborne pathogen Staphylococcus aureus from food-processing facilities. The effectiveness of 19 essential oils against planktonic cells of S. aureus was firstly assessed by minimal inhibitory concentration. Planktonic cells showed a wide variability in resistance to essential oils, with thyme oil as the most effective, followed by lemongrass oil and then vetiver oil. The eight essential oils most effective against planktonic cells were subsequently tested against 48-h-old biofilms formed on stainless steel. All essential oils reduced significantly (p < 0.01) the number of viable biofilm cells, but none of them could remove biofilms completely. Thyme and patchouli oils were the most effective, but high concentrations were needed to achieve logarithmic reductions over 4 log CFU/cm(2) after 30 min exposure. Alternatively, the use of sub-lethal doses of thyme oil allowed to slow down biofilm formation and to enhance the efficiency of thyme oil and benzalkonium chloride against biofilms. However, some cellular adaptation to thyme oil was detected. Therefore, essential oil-based treatments should be based on the rotation and combination of different essential oils or with other biocides to prevent the emergence of antimicrobial-resistant strains. PMID:25280938

  5. msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus

    PubMed Central

    Sahukhal, Gyan S.; Batte, Justin L.; Elasri, Mohamed O.

    2015-01-01

    Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm. PMID:25724778

  6. Efficacy of Combined Vancomycin and Fosfomycin against Methicillin-Resistant Staphylococcus aureus in Biofilms In Vivo

    PubMed Central

    Wang, Li; Zhang, Han-Bo; Chen, Qian; Liu, Hua; Tang, Xun; Jin, Tao; Zhu, Chong-Tao; Li, Fu-Bing; Sun, Lin-Hui; Xu, Xin-Ming; Xu, Yong-Qing

    2014-01-01

    Infection by methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. The aim of this study was to demonstrate the in vivo bactericidal effects of a combination of vancomycin (VAN) and fosfomycin (FOS) against MRSA in a rat carboxymethyl cellulose-pouch biofilm model. The results of the time-kill assay showed that the combination therapy was capable of killing at low minimal inhibitory concentrations (MIC) (½× MIC VAN +1× MIC FOS and 1× MIC VAN + 1× MIC FOS). In the in vivo study, a synergistically bactericidal effect was observed when using the combination therapy on MRSA embedded in the mature biofilm model. In comparison with the untreated control group and the groups receiving either VAN or FOS alone, the rats treated with combination therapy had lower MRSA colony counts in exudates from the pouch, lower white blood cell and neutrophil counts, and C-reactive protein (CRP) in peripheral blood. Furthermore, histological analysis of the pouch wall indicated combination therapy resulted in disappearance of biofilm-like structures, marked decrease in necrosis, and formation of granular tissue. In conclusion, the combination of VAN with FOS had a synergistic bactericidal effect on chronic MRSA infection embedded in biofilm, providing an alternative approach to treating this condition. PMID:25551618

  7. Biofilm formation by Staphylococcus aureus isolates from skin and soft tissue infections.

    PubMed

    Kwiecinski, Jakub; Kahlmeter, Gunnar; Jin, Tao

    2015-05-01

    Many diseases caused by Staphylococcus aureus are associated with biofilm formation. However, the ability of S. aureus isolates from skin and soft tissue infections to form biofilms has not yet been investigated. We tested 160 isolates from patients with various skin infections for biofilm-forming capacity in different growth media. All the isolates formed biofilms, the extent of which depended on the type of growth medium. The thickest biofilms were formed when both plasma and glucose were present in the broth; in this case, S. aureus incorporated host fibrin into the biofilm's matrix. There were no differences in the biofilm formation between isolates from different types of skin infections, except for a particularly good biofilm formation by isolates from diabetic wounds and a weaker biofilm formation by isolates from impetigo. In conclusion, biofilm formation is a universal behavior of S. aureus isolates from skin infections. In some cases, such as in diabetic wounds, a particularly strong biofilm formation most likely contributes to the chronic and recurrent character of the infection. Additionally, as S. aureus apparently uses host fibrin as part of the biofilm structure, we suggest that plasma should be included more frequently in in vitro biofilm studies. PMID:25586078

  8. Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms

    PubMed Central

    Ferreira, Inês Santos; Bettencourt, Ana F; Gonçalves, Lídia MD; Kasper, Stefanie; Bétrisey, Bertrand; Kikhney, Judith; Moter, Annette; Trampuz, Andrej; Almeida, António J

    2015-01-01

    The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was

  9. Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

    PubMed

    Ferreira, Inês Santos; Bettencourt, Ana F; Gonçalves, Lídia M D; Kasper, Stefanie; Bétrisey, Bertrand; Kikhney, Judith; Moter, Annette; Trampuz, Andrej; Almeida, António J

    2015-01-01

    The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was

  10. The Use of Commercially Available Alpha-Amylase Compounds to Inhibit and Remove Staphylococcus aureus Biofilms

    PubMed Central

    Craigen, Bradford; Dashiff, Aliza; Kadouri, Daniel E

    2011-01-01

    Staphylococcus aureus, a versatile human pathogen, is commonly associated with medical device infections. Its capacity to establish and maintain these infections is thought to be related to its ability to form adherent biofilms. In this study, commercially available α-amylase compounds from various biological sources were evaluated for their ability to reduce and prevent biofilm formation of several S. aureus isolates. Our data demonstrates that α-amylase compounds can rapidly detach biofilms of S. aureus, as well as inhibit biofilm formation. Our data also demonstrates that α-amylase compounds have an ability to reduce and disassociate S. aureus cell-aggregates grown in liquid suspension. These findings suggest that commercially available α-amylase compounds could be used in the future to control S. aureus biofilm-related infections. PMID:21760865

  11. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms

    PubMed Central

    Schwartz, Kelly; Ganesan, Mahesh; Payne, David E.; Solomon, Michael J.; Boles, Blaise R.

    2015-01-01

    Summary Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the coordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA), and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology. PMID:26365835

  12. Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa.

    PubMed

    Yang, Liang; Liu, Yang; Markussen, Trine; Høiby, Niels; Tolker-Nielsen, Tim; Molin, Søren

    2011-08-01

    Biofilm infections may not simply be the result of colonization by one bacterium, but rather the consequence of pathogenic contributions from several bacteria. Interspecies interactions of different organisms in mixed-species biofilms remain largely unexplained, but knowledge of these is very important for understanding of biofilm physiology and the treatment of biofilm-related infectious diseases. Here, we have investigated interactions of two of the major bacterial species of cystic fibrosis lung microbial communities -Pseudomonas aeruginosa and Staphylococcus aureus- when grown in co-culture biofilms. By growing co-culture biofilms of S. aureus with P. aeruginosa mutants in a flow-chamber system and observing them using confocal laser scanning microscopy, we show that wild-type P. aeruginosa PAO1 facilitates S. aureus microcolony formation. In contrast, P. aeruginosa mucA and rpoN mutants do not facilitate S. aureus microcolony formation and tend to outcompete S. aureus in co-culture biofilms. Further investigations reveal that extracellular DNA (eDNA) plays an important role in S. aureus microcolony formation and that P. aeruginosa type IV pili are required for this process, probably through their ability to bind to eDNA. Furthermore, P. aeruginosa is able to protect S. aureus against Dictyostelium discoideum phagocytosis in co-culture biofilms. PMID:21595754

  13. Ecological changes in oral microcosm biofilm during maturation.

    PubMed

    Kim, Young-Seok; Kang, Si-Mook; Lee, Eun-Song; Lee, Ji Hyun; Kim, Bo-Ra; Kim, Baek-Il

    2016-10-01

    The aim of this study was to evaluate the ecological changes in the biofilm at different stages of maturation using 16S rDNA gene amplicon sequencing and to identify correlations between red/green (R/G) fluorescence ratio and ecological changes. An oral microcosm biofilm was initiated from the saliva of a single donor and grown anaerobically for up to 10 days in basal medium mucin. Quantitative light-induced fluorescence analysis was shown that the R/G ratio of the biofilm increased consistently, but the slope rapidly decreased after six days. The bacterial compositions of 10 species also consistently changed over time. However, there was no significant correlation between each bacteria and red fluorescence. The monitoring of the maturation process of oral microcosm biofilm over 10 days revealed that the R/G ratio and the bacterial composition within biofilm consistently changed. Therefore, the R/G fluorescence ratio of biofilm may be related with its ecological change rather than specific bacteria PMID:26950795

  14. Early application of negative pressure wound therapy to acute wounds contaminated with Staphylococcus aureus: An effective approach to preventing biofilm formation

    PubMed Central

    LI, TONGTONG; ZHANG, LIHAI; HAN, LI; WANG, GUOQI; YIN, PENG; LI, ZHIRUI; ZHANG, LICHENG; GUO, QI; LIU, DAOHONG; TANG, PEIFU

    2016-01-01

    Negative pressure wound therapy (NPWT) has been demonstrated to be effective at preventing biofilm-associated infections; however, its role in biofilm prevention is unknown. The present study evaluated the effect of NPWT on biofilm prevention when rapidly initiated following wound contamination. Full-thickness dermal wounds (8 mm) were created in rabbit ears and inoculated with green fluorescent protein-labeled Staphylococcus aureus (S. aureus). At 6 h following inoculation, continuous NPWT at −125 mmHg was initiated, with the wounds on the contralateral ear left untreated in order to serve as self-controls. S. aureus rapidly formed mature biofilms in the wound beds post-inoculation, with a persistent bacterial burden of ~105−107 colony-forming units (CFUs)/wound and impaired wound healing. Compared with the untreated group, NPWT resulted in a significant reduction in biofilm matrix, which was verified by scanning electron microscopy and epifluorescence. A reduction in bacterial counts followed (P<0.05) with ~103 CFUs/wound on postoperative day 13 and improvement in all healing parameters (P<0.05) relative to control wounds. The results of the present investigation suggest that NPWT is an effective strategy to impeding the formation of S. aureus wound biofilms when initiated rapidly following bacterial contamination. The early application of NPWT, aimed at biofilm prevention, may improve wound care. PMID:26997991

  15. Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

    PubMed Central

    Wu, Julie A.; Kusuma, Caroline; Mond, James J.; Kokai-Kun, John F.

    2003-01-01

    Staphylococci often form biofilms, sessile communities of microcolonies encased in an extracellular matrix that adhere to biomedical implants or damaged tissue. Infections associated with biofilms are difficult to treat, and it is estimated that sessile bacteria in biofilms are 1,000 to 1,500 times more resistant to antibiotics than their planktonic counterparts. This antibiotic resistance of biofilms often leads to the failure of conventional antibiotic therapy and necessitates the removal of infected devices. Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan. Lysostaphin kills Staphylococcus aureus within minutes (MIC at which 90% of the strains are inhibited [MIC90], 0.001 to 0.064 μg/ml) and is also effective against Staphylococcus epidermidis at higher concentrations (MIC90, 12.5 to 64 μg/ml). The activity of lysostaphin against staphylococci present in biofilms compared to those of other antibiotics was, however, never explored. Surprisingly, lysostaphin not only killed S. aureus in biofilms but also disrupted the extracellular matrix of S. aureus biofilms in vitro on plastic and glass surfaces at concentrations as low as 1 μg/ml. Scanning electron microscopy confirmed that lysostaphin eradicated both the sessile cells and the extracellular matrix of the biofilm. This disruption of S. aureus biofilms was specific for lysostaphin-sensitive S. aureus, as biofilms of lysostaphin-resistant S. aureus were not affected. High concentrations of oxacillin (400 μg/ml), vancomycin (800 μg/ml), and clindamycin (800 μg/ml) had no effect on the established S. aureus biofilms in this system, even after 24 h. Higher concentrations of lysostaphin also disrupted S. epidermidis biofilms. PMID:14576095

  16. Streptomyces-derived actinomycin D inhibits biofilm formation by Staphylococcus aureus and its hemolytic activity.

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Lee, Kayeon; Kim, Chang-Jin; Park, Dong-Jin; Ju, Yoonjung; Lee, Jae-Chan; Wood, Thomas K; Lee, Jintae

    2016-01-01

    Staphylococcus aureus is a versatile human pathogen that produces diverse virulence factors, and its biofilm cells are difficult to eradicate due to their inherent ability to tolerate antibiotics. The anti-biofilm activities of the spent media of 252 diverse endophytic microorganisms were investigated using three S. aureus strains. An attempt was made to identify anti-biofilm compounds in active spent media and to assess their anti-hemolytic activities and hydrophobicities in order to investigate action mechanisms. Unlike other antibiotics, actinomycin D (0.5 μg ml(-1)) from Streptomyces parvulus significantly inhibited biofilm formation by all three S. aureus strains. Actinomycin D inhibited slime production in S. aureus and it inhibited hemolysis by S. aureus and caused S. aureus cells to become less hydrophobic, thus supporting its anti-biofilm effect. In addition, surface coatings containing actinomycin D prevented S. aureus biofilm formation on glass surfaces. Given these results, FDA-approved actinomycin D warrants further attention as a potential antivirulence agent against S. aureus infections. PMID:26785934

  17. Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

    PubMed Central

    Na, Manli; Jarneborn, Anders; Jacobsson, Gunnar; Peetermans, Marijke; Verhamme, Peter

    2015-01-01

    Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices. PMID:26519394

  18. Filaments in curved flow: Rapid formation of Staphylococcus aureus biofilm streamers

    NASA Astrophysics Data System (ADS)

    Kim, Min Young; Drescher, Knut; Pak, On Shun; Bassler, Bonnie L.; Stone, Howard A.

    2014-03-01

    Biofilms are surface-associated conglomerates of bacteria that are highly resistant to antibiotics. These bacterial communities can cause chronic infections in humans by colonizing, for example, medical implants, heart valves, or lungs. Staphylococcus aureus, a notorious human pathogen, causes some of the most common biofilm-related infections. Despite the clinical importance of S. aureus biofilms, it remains mostly unknown how physical effects, in particular flow, and surface structure influence biofilm dynamics. Here we use model microfluidic systems to investigate how environmental factors, such as surface geometry, surface chemistry, and fluid flow affect biofilm development in S. aureus.We discovered that S. aureus rapidly forms flow-induced, filamentous biofilm streamers, and furthermore if surfaces are coated with human blood plasma, streamers appear within minutes and clog the channels more rapidly than if the channels are uncoated. To understand how biofilm streamer filaments reorient in curved flow to bridge the distances between corners, we developed a mathematical model based on resistive force theory and slender filaments. Understanding physical aspects of biofilm formation in S. aureus may lead to new approaches for interrupting biofilm formation of this pathogen.

  19. Filaments in curved streamlines: rapid formation of Staphylococcus aureus biofilm streamers

    NASA Astrophysics Data System (ADS)

    Kim, Minyoung Kevin; Drescher, Knut; Pak, On Shun; Bassler, Bonnie L.; Stone, Howard A.

    2014-06-01

    Biofilms are surface-associated conglomerates of bacteria that are highly resistant to antibiotics. These bacterial communities can cause chronic infections in humans by colonizing, for example, medical implants, heart valves, or lungs. Staphylococcus aureus, a notorious human pathogen, causes some of the most common biofilm-related infections. Despite the clinical importance of S. aureus biofilms, it remains mostly unknown how physical effects, in particular flow, and surface structure influence biofilm dynamics. Here we use model microfluidic systems to investigate how environmental factors, such as surface geometry, surface chemistry, and fluid flow affect biofilm development of S. aureus. We discovered that S. aureus rapidly forms flow-induced, filamentous biofilm streamers, and furthermore if surfaces are coated with human blood plasma, streamers appear within minutes and clog the channels more rapidly than if the channels are uncoated. To understand how biofilm streamer filaments reorient in flows with curved streamlines to bridge the distances between corners, we developed a mathematical model based on resistive force theory of slender filaments. Understanding physical aspects of biofilm formation of S. aureus may lead to new approaches for interrupting biofilm formation of this pathogen.

  20. Prostaglandin E2 from Candida albicans Stimulates the Growth of Staphylococcus aureus in Mixed Biofilms

    PubMed Central

    Krause, Jan; Geginat, Gernot; Tammer, Ina

    2015-01-01

    Background Previous studies showed that Staphylococcus aureus and Candida albicans interact synergistically in dual species biofilms resulting in enhanced mortality in animal models. Methodology/Principal Findings The aim of the current study was to test possible candidate molecules which might mediate this synergistic interaction in an in vitro model of mixed biofilms, such as farnesol, tyrosol and prostaglandin (PG) E2. In mono-microbial and dual biofilms of C.albicans wild type strains PGE2 levels between 25 and 250 pg/mL were measured. Similar concentrations of purified PGE2 significantly enhanced S.aureus biofilm formation in a mode comparable to that observed in dual species biofilms. Supernatants of the null mutant deficient in PGE2 production did not stimulate the proliferation of S.aureus and the addition of the cyclooxygenase inhibitor indomethacin blocked the S.aureus biofilm formation in a dose-dependent manner. Additionally, S. aureus biofilm formation was boosted by low and inhibited by high farnesol concentrations. Supernatants of the farnesol-deficient C. albicans ATCC10231 strain significantly enhanced the biofilm formation of S. aureus but at a lower level than the farnesol producer SC5314. However, C. albicans ATCC10231 also produced PGE2 but amounts were significantly lower compared to SC5314. Conclusion/Significance In conclision, we identified C. albicans PGE2 as a key molecule stimulating the growth and biofilm formation of S. aureus in dual S. aureus/C. albicans biofilms, although C. albicans derived farnesol, but not tyrosol, may also contribute to this effect but to a lesser extent. PMID:26262843

  1. Nuclease Modulates Biofilm Formation in Community-Associated Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Kiedrowski, Megan R.; Kavanaugh, Jeffrey S.; Malone, Cheryl L.; Mootz, Joe M.; Voyich, Jovanka M.; Smeltzer, Mark S.; Bayles, Kenneth W.; Horswill, Alexander R.

    2011-01-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation. PMID:22096493

  2. A low molecular weight component of serum inhibits biofilm formation in Staphylococcus aureus

    PubMed Central

    Abraham, Nabil; Jefferson, Kimberly K.

    2010-01-01

    Staphylococcus aureus has a variety of genes that can influence the process of biofilm formation. The ability to establish a biofilm is an important virulence factor for this pathogen, and yet, the regulation of this process in vivo is not well understood. S. aureus can form biofilms on intravenous catheters and this process plays a key role in the pathogenesis of catheter infections. In order to investigate whether or not serum is conducive to the process of biofilm formation, we grew S. aureus in serum and analyzed biofilm thickness and expression of biofilm-related genes. Whereas serum supported planktonic bacterial growth, it was a potent inhibitor of biofilm formation. The inhibitory serum component had a molecular weight less than 3,000 kDa. The component was protease-resistant and heat stable. The serum component induced a significant increase in the transcription of the intercellular adhesin gene icaA and the fibronectin binding protein gene fnbA. Transcription of other biofilm-related genes were affected in a strain-dependent manner. These results reveal that serum inhibits biofilm formation despite the fact that biofilms form on intravenous catheters. This may suggest that in vivo, biofilm formation is “selected for” by the force of blood flow and/or immune pressure rather than “induced” by serum. PMID:20673798

  3. Analysis of Bacterial Biofilms on a Cochlear Implant Following Methicillin-Resistant Staphylococcus Aureus Infection

    PubMed Central

    An, Yun Suk; Choi, June; Song, Jae Jun; Chae, Sung Won; Jung, Hak Hyun

    2015-01-01

    To demonstrate biofilm formations on a cochlear implant magnet of a pediatric patient suffering from a methicillin-resistant Staphylococcus aureus (MRSA) infection. The appearance of biofilm colonies was analyzed on different magnet sections. The appearance of MRSA biofilms on the surface of an explanted cochlear implant was analyzed by scanning electron microscopy (SEM), focusing on the pattern of extracellular polymeric substances (EPS) within the biofilms. SEM revealed unique biofilms with a three-dimensional EPS complex and tower-like formations. Biofilm configurations changed from the margin to the center of the magnet. Biofilms were solitary and scattered at the margin; large and plate-like in the center; and stacked in layers, forming towers and water channels, in the middle region. After a MRSA infection, biofilm formations were observed on the surface of a magnet. Bacterial biofilms provide optimal conditions for bacterial growth and antibiotic resistance and can cause intractable infections that lead to device failure. PMID:26771017

  4. Vancomycin and maltodextrin affect structure and activity of Staphylococcus aureus biofilms.

    PubMed

    Kiamco, Mia Mae; Atci, Erhan; Khan, Qaiser Farid; Mohamed, Abdelrhman; Renslow, Ryan S; Abu-Lail, Nehal; Fransson, Boel A; Call, Douglas R; Beyenal, Haluk

    2015-12-01

    Hyperosmotic agents such as maltodextrin negatively impact bacterial growth through osmotic stress without contributing to drug resistance. We hypothesized that a combination of maltodextrin (osmotic agent) and vancomycin (antibiotic) would be more effective against Staphylococcus aureus biofilms than either alone. To test our hypothesis, S. aureus was grown in a flat plate flow cell reactor. Confocal laser scanning microscopy images were analyzed to quantify changes in biofilm structure. We used dissolved oxygen microelectrodes to quantify how vancomycin and maltodextrin affected the respiration rate and oxygen penetration into the biofilm. We found that treatment with vancomycin or maltodextrin altered biofilm structure. The effect on the structure was significant when they were used simultaneously to treat S. aureus biofilms. In addition, vancomycin treatment increased the oxygen respiration rate, while maltodextrin treatment caused an increase and then a decrease. An increased maltodextrin concentration decreased the diffusivity of the antibiotic. Overall, we conclude that (1) an increased maltodextrin concentration decreases vancomycin diffusion but increases the osmotic effect, leading to the optimum treatment condition, and (2) the combination of vancomycin and maltodextrin is more effective against S. aureus biofilms than either alone. Vancomycin and maltodextrin act together to increase the effectiveness of treatment against S. aureus biofilm growth. PMID:26084588

  5. Biofilm-producing ability of Staphylococcus aureus isolates from Brazilian dairy farms.

    PubMed

    Lee, S H I; Mangolin, B L C; Gonçalves, J L; Neeff, D V; Silva, M P; Cruz, A G; Oliveira, C A F

    2014-03-01

    This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n=220); milk from bulk tank (n=120); surfaces of milking machines and utensils (n=389); and milk handlers (n=120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied. PMID:24440248

  6. Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1.

    PubMed

    Nithya, Chari; Begum, Mansur Farzana; Pandian, Shunmugiah Karutha

    2010-09-01

    According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 microg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70-74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat. PMID:20665017

  7. Evidence for icaADBC-Independent Biofilm Development Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates

    PubMed Central

    Fitzpatrick, Fidelma; Humphreys, Hilary; O'Gara, James P.

    2005-01-01

    Synthesis of a polysaccharide adhesin by icaADBC-encoded enzymes is currently the best-understood mechanism of staphylococcal biofilm development. In four methicillin-resistant Staphylococcus aureus isolates, environmental activation of icaADBC did not always correlate with increased biofilm production. Moreover, glucose-mediated biofilm development in these isolates was icaADBC independent. Apparently, an environmentally regulated, ica-independent mechanism(s) of biofilm development exists in S. aureus clinical isolates. PMID:15815035

  8. Combined Use of Bacteriophage K and a Novel Bacteriophage To Reduce Staphylococcus aureus Biofilm Formation

    PubMed Central

    Alves, D. R.; Gaudion, A.; Bean, J. E.; Perez Esteban, P.; Arnot, T. C.; Harper, D. R.; Kot, W.; Hansen, L. H.; Enright, M. C.

    2014-01-01

    Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms. PMID:25149517

  9. Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources.

    PubMed

    Oniciuc, Elena-Alexandra; Cerca, Nuno; Nicolau, Anca I

    2016-01-01

    Sixteen Staphylococcus aureus isolates originating from foods (eight from dairy products, five from fish and fish products and three from meat and meat products) were evaluated regarding their biofilms formation ability. Six strains (E2, E6, E8, E10, E16, and E23) distinguished as strong biofilm formers, either in standard Tryptic Soy Broth or in Tryptic Soy Broth supplemented with 0.4% glucose or with 4% NaCl. The composition of the biofilms formed by these S. aureus strains on polystyrene surfaces was first inferred using enzymatic and chemical treatments. Later on, biofilms were characterized by confocal laser scanning microscope (CLSM). Our experiments proved that protein-based matrices are of prime importance for the structure of biofilms formed by S. aureus strains isolated from food sources. These biofilm matrix compositions are similar to those put into evidence for coagulase negative staphylococci. This is a new finding having in view that scientific literature mentions exopolysaccharide abundance in biofilms produced by clinical isolates and food processing environment isolates of S. aureus. PMID:27065962

  10. Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources

    PubMed Central

    Oniciuc, Elena-Alexandra; Cerca, Nuno; Nicolau, Anca I.

    2016-01-01

    Sixteen Staphylococcus aureus isolates originating from foods (eight from dairy products, five from fish and fish products and three from meat and meat products) were evaluated regarding their biofilms formation ability. Six strains (E2, E6, E8, E10, E16, and E23) distinguished as strong biofilm formers, either in standard Tryptic Soy Broth or in Tryptic Soy Broth supplemented with 0.4% glucose or with 4% NaCl. The composition of the biofilms formed by these S. aureus strains on polystyrene surfaces was first inferred using enzymatic and chemical treatments. Later on, biofilms were characterized by confocal laser scanning microscope (CLSM). Our experiments proved that protein-based matrices are of prime importance for the structure of biofilms formed by S. aureus strains isolated from food sources. These biofilm matrix compositions are similar to those put into evidence for coagulase negative staphylococci. This is a new finding having in view that scientific literature mentions exopolysaccharide abundance in biofilms produced by clinical isolates and food processing environment isolates of S. aureus. PMID:27065962

  11. An Optimized Lock Solution Containing Micafungin, Ethanol and Doxycycline Inhibits Candida albicans and Mixed C. albicans – Staphyloccoccus aureus Biofilms

    PubMed Central

    Lown, Livia; Peters, Brian M.; Walraven, Carla J.; Noverr, Mairi C.; Lee, Samuel A.

    2016-01-01

    Candida albicans is a major cause of catheter-related bloodstream infections and is associated with high morbidity and mortality. Due to the propensity of C. albicans to form drug-resistant biofilms, the current standard of care includes catheter removal; however, reinsertion may be technically challenging or risky. Prolonged exposure of an antifungal lock solution within the catheter in conjunction with systemic therapy has been experimentally attempted for catheter salvage. Previously, we demonstrated excellent in vitro activity of micafungin, ethanol, and high-dose doxycycline as single agents for prevention and treatment of C. albicans biofilms. Thus, we sought to investigate optimal combinations of micafungin, ethanol, and/or doxycycline as a lock solution. We performed two- and three-drug checkerboard assays to determine the in vitro activity of pairwise or three agents in combination for prevention or treatment of C. albicans biofilms. Optimal lock solutions were tested for activity against C. albicans clinical isolates, reference strains and polymicrobial C. albicans-S. aureus biofilms. A solution containing 20% (v/v) ethanol, 0.01565 μg/mL micafungin, and 800 μg/mL doxycycline demonstrated a reduction of 98% metabolic activity and no fungal regrowth when used to prevent fungal biofilm formation; however there was no advantage over 20% ethanol alone. This solution was also successful in inhibiting the regrowth of C. albicans from mature polymicrobial biofilms, although it was not fully bactericidal. Solutions containing 5% ethanol with low concentrations of micafungin and doxycycline demonstrated synergistic activity when used to prevent monomicrobial C. albicans biofilm formation. A combined solution of micafungin, ethanol and doxycycline is highly effective for the prevention of C. albicans biofilm formation but did not demonstrate an advantage over 20% ethanol alone in these studies. PMID:27428310

  12. The use of desiccation to treat Staphylococcus aureus biofilm-infected wounds.

    PubMed

    Park, Eugene; Long, Sarah A; Seth, Akhil K; Geringer, Matthew; Xu, Wei; Chavez-Munoz, Claudia; Leung, Kai; Hong, Seok Jong; Galiano, Robert D; Mustoe, Thomas A

    2016-03-01

    Chronic wounds colonized with biofilm present a major burden to our healthcare system. While the current paradigm for wound healing is to maintain a moist environment, we sought to evaluate the effects of desiccation, and the ability of honey to desiccate wounds, on wound healing characteristics in Staphylococcus aureus biofilm wounds. In vivo biofilm wound healing after exposure to open-air desiccation, honey, molasses, and saline was analyzed using a rabbit ear model of S. aureus biofilm wounds previously developed by our group. Wound morphology was examined using scanning electron microscopy and granulation tissue deposition was measured using light microscopy with hematoxylin and eosin staining. Viable bacterial counts in rabbit ear biofilm wounds and scabs were measured using a drop dilution method. In vitro S. aureus growth curves were established using tryptic soy broth containing honey and glycerol. Gene expression analysis of rabbit ear wounds was performed using reverse transcription quantitative PCR. Rabbit ear S. aureus biofilm wounds exposed to open-air desiccation, honey, and molasses developed a dry scab, which displaced the majority of biofilm bacteria off of the wound bed. Wounds treated with open-air desiccation, honey, and molasses expressed lower levels of the inflammatory markers tumor necrosis factor-α and interleukin-1β at postoperative day 12 compared with wounds treated with saline, and had increased levels of granulation tissue formation. In vitro growth of S. aureus in tryptic soy broth was inhibited by the presence of honey to a greater extent than by the presence of osmolality-matched glycerol. Desiccation of chronic wounds colonized with biofilm via exposure to open air or honey leads to improved wound healing by decreasing bacterial burden and inflammation, and increasing granulation tissue formation. The ability of honey to help heal chronic wounds is at least in part due to its ability to desiccate bacterial biofilm, but other

  13. d-Amino Acids Do Not Inhibit Biofilm Formation in Staphylococcus aureus

    PubMed Central

    Sarkar, Sourav; Pires, Marcos M.

    2015-01-01

    Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation. PMID:25658642

  14. d-Amino acids do not inhibit biofilm formation in Staphylococcus aureus.

    PubMed

    Sarkar, Sourav; Pires, Marcos M

    2015-01-01

    Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation. PMID:25658642

  15. Inhibitory Effects of d-Amino Acids on Staphylococcus aureus Biofilm Development ▿ †

    PubMed Central

    Hochbaum, Allon I.; Kolodkin-Gal, Ilana; Foulston, Lucy; Kolter, Roberto; Aizenberg, Joanna; Losick, Richard

    2011-01-01

    Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in Bacillus subtilis is certain d-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix. d-Amino acids also prevent biofilm formation by the related Gram-positive bacterium Staphylococcus aureus. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how d-amino acids prevent biofilm formation by S. aureus. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates. d-Amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that d-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that d-amino acids act in an analogous manner to prevent biofilm development in B. subtilis and S. aureus. Finally, to investigate the potential utility of d-amino acids in preventing device-related infections, we have shown that surfaces impregnated with d-amino acids were effective in preventing biofilm growth. PMID:21856845

  16. Effects of Low-Dose Amoxicillin on Staphylococcus aureus USA300 Biofilms.

    PubMed

    Mlynek, Kevin D; Callahan, Mary T; Shimkevitch, Anton V; Farmer, Jackson T; Endres, Jennifer L; Marchand, Mélodie; Bayles, Kenneth W; Horswill, Alexander R; Kaplan, Jeffrey B

    2016-05-01

    Previous studies showed that sub-MIC levels of β-lactam antibiotics stimulate biofilm formation in most methicillin-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated this process by measuring the effects of sub-MIC amoxicillin on biofilm formation by the epidemic community-associated MRSA strain USA300. We found that sub-MIC amoxicillin increased the ability of USA300 cells to attach to surfaces and form biofilms under both static and flow conditions. We also found that USA300 biofilms cultured in sub-MIC amoxicillin were thicker, contained more pillar and channel structures, and were less porous than biofilms cultured without antibiotic. Biofilm formation in sub-MIC amoxicillin correlated with the production of extracellular DNA (eDNA). However, eDNA released by amoxicillin-induced cell lysis alone was evidently not sufficient to stimulate biofilm. Sub-MIC levels of two other cell wall-active agents with different mechanisms of action-d-cycloserine and fosfomycin-also stimulated eDNA-dependent biofilm, suggesting that biofilm formation may be a mechanistic adaptation to cell wall stress. Screening a USA300 mariner transposon library for mutants deficient in biofilm formation in sub-MIC amoxicillin identified numerous known mediators of S. aureus β-lactam resistance and biofilm formation, as well as novel genes not previously associated with these phenotypes. Our results link cell wall stress and biofilm formation in MRSA and suggest that eDNA-dependent biofilm formation by strain USA300 in low-dose amoxicillin is an inducible phenotype that can be used to identify novel genes impacting MRSA β-lactam resistance and biofilm formation. PMID:26856828

  17. Random peptide mixtures inhibit and eradicate methicillin-resistant Staphylococcus aureus biofilms.

    PubMed

    Stern, Tal; Zelinger, Einat; Hayouka, Zvi

    2016-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a biofilm-forming pathogen that can cause serious health complications in humans, ranging from minor to life-threatening infections. The challenge of successfully combating biofilms requires the discovery of compounds with a novel mode of action. We have recently developed sequence-random hydrophobic-cationic peptides that display a broad antibacterial activity. In the current study we show that our novel compounds are capable of controlling and managing MRSA biofilms and might be used as lead biofilm inhibitor candidates for further studies. PMID:27161246

  18. Bdellovibrio bacteriovorus Inhibits Staphylococcus aureus Biofilm Formation and Invasion into Human Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Monnappa, Ajay K.; Dwidar, Mohammed; Seo, Jeong Kon; Hur, Jin-Hoe; Mitchell, Robert J.

    2014-01-01

    Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks many Gram-negative human pathogens. A serious drawback of this strain, however, is its ineffectiveness against Gram-positive strains, such as the human pathogen Staphylococcus aureus. Here we demonstrate that the extracellular proteases produced by a host-independent B. bacteriovorus (HIB) effectively degrade/inhibit the formation of S. aureus biofilms and reduce its virulence. A 10% addition of HIB supernatant caused a 75% or greater reduction in S. aureus biofilm formation as well as 75% dispersal of pre-formed biofilms. LC-MS-MS analyses identified various B. bacteriovorus proteases within the supernatant, including the serine proteases Bd2269 and Bd2321. Tests with AEBSF confirmed that serine proteases were active in the supernatant and that they impacted S. aureus biofilm formation. The supernatant also possessed a slight DNAse activity. Furthermore, treatment of planktonic S. aureus with the supernatant diminished its ability to invade MCF-10a epithelial cells by 5-fold but did not affect the MCF-10a viability. In conclusion, this study illustrates the hitherto unknown ability of B. bacteriovorus to disperse Gram-positive pathogenic biofilms and mitigate their virulence.

  19. Bdellovibrio bacteriovorus Inhibits Staphylococcus aureus Biofilm Formation and Invasion into Human Epithelial Cells

    PubMed Central

    Monnappa, Ajay K.; Dwidar, Mohammed; Seo, Jeong Kon; Hur, Jin-Hoe; Mitchell, Robert J.

    2014-01-01

    Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks many Gram-negative human pathogens. A serious drawback of this strain, however, is its ineffectiveness against Gram-positive strains, such as the human pathogen Staphylococcus aureus. Here we demonstrate that the extracellular proteases produced by a host-independent B. bacteriovorus (HIB) effectively degrade/inhibit the formation of S. aureus biofilms and reduce its virulence. A 10% addition of HIB supernatant caused a 75% or greater reduction in S. aureus biofilm formation as well as 75% dispersal of pre-formed biofilms. LC-MS-MS analyses identified various B. bacteriovorus proteases within the supernatant, including the serine proteases Bd2269 and Bd2321. Tests with AEBSF confirmed that serine proteases were active in the supernatant and that they impacted S. aureus biofilm formation. The supernatant also possessed a slight DNAse activity. Furthermore, treatment of planktonic S. aureus with the supernatant diminished its ability to invade MCF-10a epithelial cells by 5-fold but did not affect the MCF-10a viability. In conclusion, this study illustrates the hitherto unknown ability of B. bacteriovorus to disperse Gram-positive pathogenic biofilms and mitigate their virulence. PMID:24448451

  20. Infectious Dose Dictates the Host Response during Staphylococcus aureus Orthopedic-Implant Biofilm Infection.

    PubMed

    Vidlak, Debbie; Kielian, Tammy

    2016-07-01

    Staphylococcus aureus is a leading cause of prosthetic joint infections (PJIs) that are typified by biofilm formation. Given the diversity of S. aureus strains and their propensity to cause community- or hospital-acquired infections, we investigated whether the immune response and biofilm growth during PJI were conserved among distinct S. aureus clinical isolates. Three S. aureus strains representing USA200 (UAMS-1), USA300 (LAC), and USA400 (MW2) lineages were equally effective at biofilm formation in a mouse model of PJI and elicited similar leukocyte infiltrates and cytokine/chemokine profiles. Another factor that may influence the course of PJI is infectious dose. In particular, higher bacterial inocula could accelerate biofilm formation and alter the immune response, making it difficult to discern underlying pathophysiological mechanisms. To address this issue, we compared the effects of two bacterial doses (10(3) or 10(5) CFU) on inflammatory responses in interleukin-12p40 (IL-12p40) knockout mice that were previously shown to have reduced myeloid-derived suppressor cell recruitment concomitant with bacterial clearance after low-dose challenge (10(3) CFU). Increasing the infectious dose of LAC to 10(5) CFU negated these differences in IL-12p40 knockout animals, demonstrating the importance of bacterial inoculum on infection outcome. Collectively, these observations highlight the importance of considering infectious dose when assessing immune responsiveness, whereas biofilm formation during PJI is conserved among clinical isolates commonly used in mouse S. aureus infection models. PMID:27091926

  1. Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

    PubMed

    Diaz De Rienzo, M A; Stevenson, P S; Marchant, R; Banat, I M

    2016-07-01

    Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds. PMID:26825819

  2. Individual Constituents from Essential Oils Inhibit Biofilm Mass Production by Multi-Drug Resistant Staphylococcus aureus.

    PubMed

    Espina, Laura; Pagán, Rafael; López, Daniel; García-Gonzalo, Diego

    2015-01-01

    Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings. PMID:26102069

  3. Development of a Standard Test To Assess the Resistance of Staphylococcus aureus Biofilm Cells to Disinfectants

    PubMed Central

    Luppens, Suzanne B. I.; Reij, Martine W.; van der Heijden, Rob W. L.; Rombouts, Frank M.; Abee, Tjakko

    2002-01-01

    A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests. PMID:12200265

  4. Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms.

    PubMed

    Lázaro-Díez, María; Remuzgo-Martínez, Sara; Rodríguez-Mirones, Cristina; Acosta, Felix; Icardo, Jose M; Martínez-Martínez, Luis; Ramos-Vivas, José

    2016-01-01

    Ceftaroline (CPT) is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections. PMID:26800524

  5. Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms

    PubMed Central

    Rodríguez-Mirones, Cristina; Acosta, Felix; Icardo, Jose M.; Martínez-Martínez, Luis; Ramos-Vivas, José

    2016-01-01

    Ceftaroline (CPT) is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections. PMID:26800524

  6. Inhibition of Biofilm Formation by Esomeprazole in Pseudomonas aeruginosa and Staphylococcus aureus

    PubMed Central

    Singh, Vandana; Arora, Vaneet; Alam, M. Jahangir

    2012-01-01

    Staphylococcus aureus and Pseudomonas aeruginosa are common nosocomial pathogens responsible for biofilm-associated infections. Proton pump inhibitors (PPI), such as esomeprazole, may have novel antimicrobial properties. The objective of this study was to assess whether esomeprazole prevents sessile bacterial growth and biofilm formation and whether it may have synergistic killing effects with standard antibiotics. The antibiofilm activity of esomeprazole at 0.25 mM was tested against two strains each of S. aureus and P. aeruginosa. Bacterial biofilms were prepared using a commercially available 96-peg-plate Calgary biofilm device. Sessile bacterial CFU counts and biomass were assessed during 72 hours of esomeprazole exposure. The killing activities after an additional 24 hours of vancomycin (against S. aureus) and meropenem (against P. aeruginosa) treatment with or without preexposure to esomeprazole were also assessed by CFU and biomass analyses. P. aeruginosa and S. aureus strains exposed to esomeprazole displayed decreased sessile bacterial growth and biomass (P < 0.001, each parameter). After 72 h of exposure, there was a 1-log10 decrease in the CFU/ml of esomeprazole-exposed P. aeruginosa and S. aureus strains compared to controls (P < 0.001). After 72 h of exposure, measured absorbance was 100% greater in P. aeruginosa control strains than in esomeprazole-exposed strains (P < 0.001). Increased killing and decreased biomass were observed for esomeprazole-treated bacteria compared to untreated controls exposed to conventional antibiotics (P < 0.001, each parameter). Reduced biofilm growth after 24 h was visibly apparent by light micrographs for P. aeruginosa and S. aureus isolates exposed to esomeprazole compared to untreated controls. In conclusion, esomeprazole demonstrated an antibiofilm effect against biofilm-producing S. aureus and P. aeruginosa. PMID:22664967

  7. Baicalein Inhibits Staphylococcus aureus Biofilm Formation and the Quorum Sensing System In Vitro

    PubMed Central

    Wang, Ke; Hou, Changchun; Cai, Shuangqi; Huang, Yingying; Du, Zhongye; Huang, Hong; Kong, Jinliang; Chen, Yiqiang

    2016-01-01

    Biofilm formed by Staphylococcus aureus significantly enhances antibiotic resistance by inhibiting the penetration of antibiotics, resulting in an increasingly serious situation. This study aimed to assess whether baicalein can prevent Staphylococcus aureus biofilm formation and whether it may have synergistic bactericidal effects with antibiotics in vitro. To do this, we used a clinically isolated strain of Staphylococcus aureus 17546 (t037) for biofilm formation. Virulence factors were detected following treatment with baicalein, and the molecular mechanism of its antibiofilm activity was studied. Plate counting, crystal violet staining, and fluorescence microscopy revealed that 32 μg/mL and 64 μg/mL baicalein clearly inhibited 3- and 7-day biofilm formation in vitro. Moreover, colony forming unit count, confocal laser scanning microscopy, and scanning electron microscopy showed that vancomycin (VCM) and baicalein generally enhanced destruction of biofilms, while VCM alone did not. Western blotting and real-time quantitative polymerase chain reaction analyses (RTQ-PCR) confirmed that baicalein treatment reduced staphylococcal enterotoxin A (SEA) and α-hemolysin (hla) levels. Most strikingly, real-time qualitative polymerase chain reaction data demonstrated that 32 μg/mL and 64 μg/mL baicalein downregulated the quorum-sensing system regulators agrA, RNAIII, and sarA, and gene expression of ica, but 16 μg/mL baicalein had no effect. In summary, baicalein inhibited Staphylococcus aureus biofilm formation, destroyed biofilms, increased the permeability of vancomycin, reduced the production of staphylococcal enterotoxin A and α-hemolysin, and inhibited the quorum sensing system. These results support baicalein as a novel drug candidate and an effective treatment strategy for Staphylococcus aureus biofilm-associated infections. PMID:27128436

  8. Role of JAK-STAT signaling in maturation of phagosomes containing Staphylococcus aureus

    PubMed Central

    Zhu, Fei; Zhou, Yadong; Jiang, Chunxia; Zhang, Xiaobo

    2015-01-01

    Phagocytosis is a required mechanism for the defense against pathogens. Staphylococcus aureus, an important bacterial pathogen, can promptly escape from phagosomes and proliferate within the cytoplasm of host. However, the mechanism of phagocytosis against S. aureus has not been intensively investigated. In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages. Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus. Genome-wide analysis and quantitative real-time PCR indicated that the JAK-STAT pathway was activated by PG during the phagosome maturation of macrophages against S. aureus. This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation. Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages. PMID:26442670

  9. (+)-Dehydroabietic Acid, an Abietane-Type Diterpene, Inhibits Staphylococcus aureus Biofilms in Vitro

    PubMed Central

    Fallarero, Adyary; Skogman, Malena; Kujala, Janni; Rajaratnam, Mohanathas; Moreira, Vânia M.; Yli-Kauhaluoma, Jari; Vuorela, Pia

    2013-01-01

    Potent drugs are desperately needed to counteract bacterial biofilm infections, especially those caused by gram-positive organisms, such as Staphylococcus aureus. Moreover, anti-biofilm compounds/agents that can be used as chemical tools are also needed for basic in vitro or in vivo studies aimed at exploring biofilms behavior and functionability. In this contribution, a collection of naturally-occurring abietane-type diterpenes and their derivatives was tested against S. aureus biofilms using a platform consisting of two phenotypic assays that have been previously published by our group. Three active compounds were identified: nordehydroabietylamine (1), (+)-dehydroabietic acid (2) and (+)-dehydroabietylamine (3) that prevented biofilm formation in the low micromolar range, and unlike typical antibiotics, only 2 to 4-fold higher concentrations were needed to significantly reduce viability and biomass of existing biofilms. Compound 2, (+)-dehydroabietic acid, was the most selective towards biofilm bacteria, achieving high killing efficacy (based on log Reduction values) and it was best tolerated by three different mammalian cell lines. Since (+)-dehydroabietic acid is an easily available compound, it holds great potential to be used as a molecular probe in biofilms-related studies as well as to serve as inspirational chemical model for the development of potent drug candidates. PMID:23739682

  10. Delivery of fluorophores by calcium phosphate-coated nanoliposomes and interaction with Staphylococcus aureus biofilms.

    PubMed

    Rivero Berti, Ignacio; Dell' Arciprete, María Laura; Dittler, María Laura; Miñan, Alejandro; Fernández Lorenzo de Mele, Mónica; Gonzalez, Mónica

    2016-06-01

    The delivery capacity and mechanical stability of calcium phosphate (CaP) coated 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) liposomes free and adsorbed on bacterial surface was investigated introducing either acridine orange (AO) or 5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin (TMP) in the aqueous core of the liposomes. The obtained nanomaterials were thoroughly characterized by electron and optical microscopy and by fluorescence techniques. Distribution of the AO and TMP molecules between the aqueous liposomes core and the outer solution was demonstrated by the band shifts and broadening of the excitation-emission matrices and the modified Stern-Volmer model for fluorescence quenching. In aqueous suspensions, c.a. 40% of AO was released to the outer solution while only a small percentage of TMP was observed to reach the outer liposome surface. The nanoliposomes adhesion capacity and the leaking of fluorophore molecules to Staphylococcus aureus (S. aureus) biofilms were further evaluated. A close interaction between liposomes and S. aureus biofilm was evidenced by TEM and SEM imaging. Epifluorescence experiments demonstrated that CaP-coated liposomes have good biofilm staining capability after two hours incubation of the biofilms with the liposomes, thus supporting an important release of the fluorophores when in contact with the biofilm. Altogether, the obtained results strongly suggest that CaP-coated liposomes are capable of activating drug release when in presence of S. aureus biofilms and smears. The studies herein presented, indicate that CaP-coated liposomes are potential vehicles for the selective delivery of drugs to S. aureus biofilms, as is the case of the singlet oxygen photosensitizer TMP, a well known photodynamic antibacterial agent. PMID:26954088

  11. Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus

    PubMed Central

    Cucarella, Carme; Tormo, M. Ángeles; Úbeda, Carles; Trotonda, M. Pilar; Monzón, Marta; Peris, Critòfol; Amorena, Beatriz; Lasa, Íñigo; Penadés, José R.

    2004-01-01

    Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica+ bap+), group 2 (ica+, bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus. PMID:15039341

  12. Effect of negative pressure on growth, secretion and biofilm formation of Staphylococcus aureus.

    PubMed

    Li, Tongtong; Wang, Guoqi; Yin, Peng; Li, Zhirui; Zhang, Licheng; Liu, Jianheng; Li, Ming; Zhang, Lihai; Han, Li; Tang, Peifu

    2015-10-01

    Negative pressure wound therapy (NPWT) has gained popularity in the management of contaminated wounds as an effective physical therapy, although its influence on the bacteria in the wounds remains unclear. In this study, we attempted to explore the effect of negative pressure conditions on Staphylococcus aureus, the most frequently isolated pathogen during wound infection. S. aureus was cultured in Luria-Bertani medium at subatmospheric pressure of -125 mmHg for 24 h, with the bacteria grown at ambient pressure as the control. The application of negative pressure was found to slow down the growth rate and inhibit biofilm development of S. aureus, which was confirmed by static biofilm assays. Furthermore, decreases in the total amount of virulence factors and biofilm components were observed, including α-hemolysin, extracellular adherence protein, polysaccharide intercellular adhesin and extracellular DNA. With quantitative RT-PCR analysis, we also revealed a significant inhibition in the transcription of virulence and regulatory genes related to wound infections and bacterial biofilms. Together, these findings indicated that negative pressure could inhibit the growth, virulence and biofilm formation of S. aureus. A topical subatmospheric pressure condition, such as NPWT, may be a potential antivirulence and antibiofilm strategy in the field of wound care. PMID:26272011

  13. Detection of genes involved in biofilm formation in Staphylococcus aureus isolates

    PubMed Central

    Nourbakhsh, Fahimeh; Namvar, Amirmorteza Ebrahimzadeh

    2016-01-01

    Staphylococcus aureus is one of the Gram-positive pathogens causing a wide range of nosocomial infections. The present study investigates genotypic and phenotypic aspects involved in biofilm formation in methicillin-resistant Staphylococcus aureus strains isolated from nosocomial infections in Isfahan. A total of 110 S. aureus strains were collected from three major hospitals in Isfahan, the center of Iran. The antibiotic resistance pattern, phenotypes, and biofilm formation genes were studied using Congo red agar (CRA) and multiplex PCR (M-PCR). We found that 103 out of 110 samples (93.6%) were MRSA. The highest frequency of resistance was found to penicillin (89%), ciprofloxacin (87.4%), and erythromycin (86.1%). Phenotypic results showed that 53.5% were high biofilm producers, while 33.3% and 13.2% were intermediate and low biofilm producers, respectively. icaC (69.3%) had the highest frequency in comparison to other intercellular adhesion (ica) genes, icaD (54.8%) was second most common. The results show that the adherence or attachment ability and biofilm production are important for enhancing virulence factors among isolates of S. aureus strains. PMID:27303652

  14. Targeting macrophage activation for the prevention and treatment of S. aureus biofilm infections†

    PubMed Central

    Hanke, Mark L.; Heim, Cortney E.; Angle, Amanda; Sanderson, Sam D.; Kielian, Tammy

    2013-01-01

    Biofilm infections often lead to significant morbidity due to their chronicity and recalcitrance to antibiotics. We have demonstrated that methicillin-resistant Staphylococcus aureus (MRSA) biofilms can evade macrophage antibacterial effector mechanisms by skewing macrophages towards an alternatively activated M2 phenotype. To overcome this immune evasion, we have utilized two complementary approaches. In the first, a proinflammatory milieu was elicited by local administration of classically-activated M1 macrophages and second, by treatment with the C5a receptor (CD88) agonist EP67, which invokes macrophage proinflammatory activity. Early administration of M1-activated macrophages or EP67 significantly attenuated biofilm formation in a mouse model of MRSA catheter-associated infection. Several proinflammatory mediators were significantly elevated in biofilm infected tissues from macrophage- and EP67-treated animals, revealing effective reprogramming of the biofilm environment to a proinflammatory milieu. A requirement for macrophage proinflammatory activity was demonstrated by the fact that transfer of MyD88-deficient macrophages had minimal impact on biofilm growth. Likewise, neutrophil administration had no effect on biofilm formation. Treatment of established biofilm infections with M1-activated macrophages also significantly reduced catheter-associated biofilm burdens compared to antibiotic treatment. Collectively, these results demonstrate that targeting macrophage proinflammatory activity can overcome the local immune inhibitory environment created during biofilm infections and represents a novel therapeutic strategy. PMID:23365077

  15. Biofilm formation on tympanostomy tubes depends on methicillin-resistant Staphylococcus aureus genetic lineage.

    PubMed

    Jotić, Ana; Božić, Dragana D; Milovanović, Jovica; Pavlović, Bojan; Ješić, Snežana; Pelemiš, Mijomir; Novaković, Marko; Ćirković, Ivana

    2016-03-01

    Bacterial biofilm formation has been implicated in the high incidence of persistent otorrhoea after tympanostomy tube insertion. The aim of the study was to investigate whether biofilm formation on tympanostomy tubes depends on the genetic profile of methicillin-resistant Staphylococcus aureus (MRSA) strains. Capacity of biofilm formation on fluoroplastic tympanostomy tubes (TTs) was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on TTs. Tested MRSA strains were classified into SCCmec type I (36.7 %), III (23.3 %), IV (26.7 %) and V (13.3 %), agr type I (50 %), II (36.7 %) and III (13.3 %), and 5 clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on TTs was as following: 13.3 % of strains belonged to the category of no biofilm producers, 50 % to the category of weak biofilm producers and 36.7 % to moderate biofilm producers. There was a statistically significant difference between CC, SCCmec and agr types and the category of biofilm production on TTs tubes (p < 0.001): CC5, SCCmecI type and agrII type with a moderate amount of biofilm, and CC8 and agrI type with a low amount of biofilm. Biofilm formation by MRSA on TTs is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related post-tympanostomy tube otorrhea. PMID:25796207

  16. Anti-biofilm agents: recent breakthrough against multi-drug resistant Staphylococcus aureus.

    PubMed

    Chung, Pooi Y; Toh, Yien S

    2014-04-01

    Staphylococcus aureus is a Gram-positive pathogen that causes potentially life-threatening nosocomial- and community-acquired infections, such as osteomyelitis and endocarditis. Staphylococcus aureus has the ability to form multicellular, surface-adherent communities called biofilms, which enables it to survive in various sources of stress, including antibiotics, nutrient limitations, heat shock, and immune responses. Biofilm-forming capacity is now recognized as an important virulence determinant in the development of staphylococcal device-related infections. In light of the projected increase in the numbers of elderly patients who will require semi-permanent indwelling medical devices such as artificial knees and hips, we can anticipate an expanded need for new agents and treatment options to manage biofilm-associated infections in an expanding at-risk population. With better understanding of staphylococcal biofilm formation and growth, novel strategies that target biofilm-associated infections caused by S. aureus have recently been described and seem promising as future anti-biofilm therapies. PMID:24453168

  17. Differential protection from tobramycin by extracellular polymeric substances from Acinetobacter baumannii and Staphylococcus aureus biofilms.

    PubMed

    Davenport, Emily K; Call, Douglas R; Beyenal, Haluk

    2014-08-01

    We investigated biofilms of two pathogens, Acinetobacter baumannii and Staphylococcus aureus, to characterize mechanisms by which the extracellular polymeric substance (EPS) found in biofilms can protect bacteria against tobramycin exposure. To do so, it is critical to study EPS-antibiotic interactions in a homogeneous environment without mass transfer limitations. Consequently, we developed a method to grow biofilms, harvest EPS, and then augment planktonic cultures with isolated EPS and tobramycin. We demonstrated that planktonic cultures respond differently to being treated with different types of EPS (A. baumannii versus S. aureus) in the presence of tobramycin. By harvesting EPS from the biofilms, we found that A. baumannii EPS acts as a "universal protector" by inhibiting tobramycin activity against bacterial cells regardless of species; S. aureus EPS did not show any protective ability in cell cultures. Adding Mg(2+) or Ca(2+) reduced the protective effect of A. baumannii EPS. Finally, when we selectively digested the proteins or DNA of the EPS, we found that the protective ability did not change, suggesting that neither has a significant role in protection. To the best of our knowledge, this is the first study that demonstrates how EPS protects pathogens against antibiotics in a homogeneous system without mass transfer limitations. Our results suggest that EPS protects biofilm communities, in part, by adsorbing antibiotics near the surface. This may limit antibiotic diffusion to the bottom of the biofilms but is not likely to be the only mechanism of protection. PMID:24913166

  18. Differential Protection from Tobramycin by Extracellular Polymeric Substances from Acinetobacter baumannii and Staphylococcus aureus Biofilms

    PubMed Central

    Davenport, Emily K.; Call, Douglas R.

    2014-01-01

    We investigated biofilms of two pathogens, Acinetobacter baumannii and Staphylococcus aureus, to characterize mechanisms by which the extracellular polymeric substance (EPS) found in biofilms can protect bacteria against tobramycin exposure. To do so, it is critical to study EPS-antibiotic interactions in a homogeneous environment without mass transfer limitations. Consequently, we developed a method to grow biofilms, harvest EPS, and then augment planktonic cultures with isolated EPS and tobramycin. We demonstrated that planktonic cultures respond differently to being treated with different types of EPS (A. baumannii versus S. aureus) in the presence of tobramycin. By harvesting EPS from the biofilms, we found that A. baumannii EPS acts as a “universal protector” by inhibiting tobramycin activity against bacterial cells regardless of species; S. aureus EPS did not show any protective ability in cell cultures. Adding Mg2+ or Ca2+ reduced the protective effect of A. baumannii EPS. Finally, when we selectively digested the proteins or DNA of the EPS, we found that the protective ability did not change, suggesting that neither has a significant role in protection. To the best of our knowledge, this is the first study that demonstrates how EPS protects pathogens against antibiotics in a homogeneous system without mass transfer limitations. Our results suggest that EPS protects biofilm communities, in part, by adsorbing antibiotics near the surface. This may limit antibiotic diffusion to the bottom of the biofilms but is not likely to be the only mechanism of protection. PMID:24913166

  19. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma.

    PubMed

    Watters, Chase M; Burton, Tarea; Kirui, Dickson K; Millenbaugh, Nancy J

    2016-01-01

    Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%-50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections. PMID:27175088

  20. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma

    PubMed Central

    Watters, Chase M; Burton, Tarea; Kirui, Dickson K; Millenbaugh, Nancy J

    2016-01-01

    Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%–50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections. PMID:27175088

  1. Novel application for the prevention and treatment of Staphylococcus aureus biofilm formation

    NASA Astrophysics Data System (ADS)

    Traba, Christian

    Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this dissertation, the application of plasma from two very different facets was studied. In part one, the susceptibility of pre-formed Staphylococcus aureus biofilms on biomaterials to different plasmas was investigated. It was found that the distinct chemical/physical properties of plasmas generated from oxygen, nitrogen, and argon all demonstrated very potent but very different anti-biofilm mechanisms of action. An in depth analysis of these results show: 1) different reactive species produced in each plasma demonstrate specific activity, and 2) the commonly associated etching effect could be manipulated and even controlled, depending on experimental conditions and the discharge gas. These studies provide insights into the anti-biofilm mechanisms of plasma as well as the effects of different reactive species on biofilm inactivation. Under experimental parameters, bacterial cells in Staphylococcus aureus biofilms were killed (>99.9%) by plasmas within minutes of exposure and no bacteria nor biofilm re-growth from discharge gas treated biofilms was observed throughout the life-span of the re-growth experiment. The decontamination ability of plasmas for the treatment of biofilm related infections on biomedical materials was confirmed and novel applications involving the use of low power argon and oxygen for the treatment of biofilm contaminated biomaterials and indwelling devices is proposed. The second facet of this dissertation explores the interaction between biofilm forming Staphylococcus aureus bacteria on different antibacterial/anti-biofilm surfaces. The antibiotic-free anti-fouling surfaces constructed in this study were generated from the plasma-assisted graft polymerization technique. These sophisticated surfaces were stable, biocompatible and capable of preventing biofilm formation on biomaterials and medical devices. Under

  2. Ellagic Acid Derivatives from Rubus ulmifolius Inhibit Staphylococcus aureus Biofilm Formation and Improve Response to Antibiotics

    PubMed Central

    Quave, Cassandra L.; Estévez-Carmona, Miriam; Compadre, Cesar M.; Hobby, Gerren; Hendrickson, Howard; Beenken, Karen E.; Smeltzer, Mark S.

    2012-01-01

    Background Biofilms contribute to the pathogenesis of many forms of Staphylococcus aureus infection. Treatment of these infections is complicated by intrinsic resistance to conventional antibiotics, thus creating an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections. Methodology/Principal Findings This study demonstrates that a botanical natural product composition (220D-F2) rich in ellagic acid and its derivatives can limit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility. The source of this composition is Rubus ulmifolius Schott. (Rosaceae), a plant used in complementary and alternative medicine in southern Italy for the treatment of skin and soft tissue infections. All S. aureus clonal lineages tested exhibited a reduced capacity to form a biofilm at 220D-F2 concentrations ranging from 50–200 µg/mL, which were well below the concentrations required to limit bacterial growth (530–1040 µg/mL). This limitation was therapeutically relevant in that inclusion of 220D-F2 resulted in enhanced susceptibility to the functionally-distinct antibiotics daptomycin, clindamycin and oxacillin. Testing with kidney and liver cell lines also demonstrated a lack of host cell cytotoxicity at concentrations of 220D-F2 required to achieve these effects. Conclusions/Significance These results demonstrate that extract 220D-F2 from the root of Rubus ulmifolius can be used to inhibit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility without toxic effects on normal mammalian cells. Hence, 220D-F2 is a strong candidate for development as a botanical drug for use in the prevention and treatment of S. aureus biofilm-associated infections. PMID:22242149

  3. Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci.

    PubMed

    de Oliveira, Adilson; Cataneli Pereira, Valéria; Pinheiro, Luiza; Moraes Riboli, Danilo Flávio; Benini Martins, Katheryne; Ribeiro de Souza da Cunha, Maria de Lourdes

    2016-01-01

    The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS). Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus). Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC) for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB). Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis) were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4%) S. aureus strains that were resistant to oxacillin and six (42.8%) that were resistant to erythromycin. Among the CoNS, 31 (88.6%) strains were resistant to oxacillin, 14 (40%) to erythromycin, 18 (51.4%) to gentamicin, and 8 (22.8%) to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and CoNS species

  4. Laser irradiation effect on Staphylococcus aureus and Pseudomonas aeruginosa biofilms isolated from venous leg ulcer.

    PubMed

    Baffoni, Marina; Bessa, Lucinda J; Grande, Rossella; Di Giulio, Mara; Mongelli, Matteo; Ciarelli, Antonio; Cellini, Luigina

    2012-10-01

    Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, represent a significant cause of morbidity in developed countries, predominantly in older patients. The aetiology of these wounds is probably multifactorial, but the role of bacteria in their pathogenesis is still unclear. Moreover, the presence of bacterial biofilms has been considered an important factor responsible for wounds chronicity. We aimed to investigate the laser action as a possible biofilm eradicating strategy, in order to attempt an additional treatment to antibiotic therapy to improve wound healing. In this work, the effect of near-infrared (NIR) laser was evaluated on mono and polymicrobial biofilms produced by two pathogenic bacterial strains, Staphylococcus aureus PECHA10 and Pseudomonas aeruginosa PECHA9, both isolated from a chronic venous leg ulcer. Laser effect was assessed by biomass measurement, colony forming unit count and cell viability assay. It was shown that the laser treatment has not affected the biofilms biomass neither the cell viability, although a small disruptive action was observed in the structure of all biofilms tested. A reduction on cell growth was observed in S. aureus and in polymicrobial biofilms. This work represents an initial in vitro approach to study the influence of NIR laser treatment on bacterial biofilms in order to explain its potentially advantageous effects in the healing process of chronic infected wounds. PMID:22182280

  5. Mutation of sarA in Staphylococcus aureus Limits Biofilm Formation

    PubMed Central

    Beenken, Karen E.; Blevins, Jon S.; Smeltzer, Mark S.

    2003-01-01

    Mutation of sarA resulted in a reduced capacity to form a biofilm in six of the eight Staphylococcus aureus strains we tested (UAMS-1, UAMS-601, SA113, SC-01, S6C, and DB). The exceptions were Newman, which formed a poor biofilm under all conditions, and RN6390, which consistently formed a biofilm only after mutation of agr. Mutation of agr in other strains had little impact on biofilm formation. In every strain other than Newman, including RN6390, simultaneous mutation of sarA and agr resulted in a phenotype like that observed with the sarA mutants. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. PMID:12819120

  6. Liposome containing cinnamon oil with antibacterial activity against methicillin-resistant Staphylococcus aureus biofilm.

    PubMed

    Cui, Haiying; Li, Wei; Li, Changzhu; Vittayapadung, Saritporn; Lin, Lin

    2016-01-01

    The global burden of bacterial disease remains high and is set against a backdrop of increasing antimicrobial resistance. There is a pressing need for highly effective and natural antibacterial agents. In this work, the anti-biofilm effect of cinnamon oil on methicillin-resistant Staphylococcus aureus was evaluated. Then, cinnamon oil was encapsulated in liposomes to enhance its chemical stability. The anti-biofilm activities of the liposome-encapsulated cinnamon oil against MRSA biofilms on stainless steel, gauze, nylon membrane and non-woven fabrics were evaluated by colony forming unit determination. Scanning electron microscopy and laser scanning confocal microscopy analyses were employed to observe the morphological changes in MRSA biofilms treated with the encapsulated cinnamon oil. As a natural and safe spice, the cinnamon oil exhibited a satisfactory antibacterial performance on MRSA and its biofilms. The application of liposomes further improves the stability of antimicrobial agents and extends the action time. PMID:26838161

  7. Effect of Farnesol on Staphylococcus aureus Biofilm Formation and Antimicrobial Susceptibility

    PubMed Central

    Jabra-Rizk, M. A.; Meiller, T. F.; James, C. E.; Shirtliff, M. E.

    2006-01-01

    Staphylococcus aureus is among the leading pathogens causing bloodstream infections able to form biofilms on host tissue and indwelling medical devices and to persist and cause disease. Infections caused by S. aureus are becoming more difficult to treat because of increasing resistance to antibiotics. In a biofilm environment particularly, microbes exhibit enhanced resistance to antimicrobial agents. Recently, farnesol was described as a quorum-sensing molecule with possible antimicrobial properties. In this study, the effect of farnesol on methicillin-resistant and -susceptible strains of S. aureus was investigated. With viability assays, biofilm formation assessment, and ethidium bromide uptake testing, farnesol was shown to inhibit biofilm formation and compromise cell membrane integrity. The ability of farnesol to sensitize S. aureus to antimicrobials was assessed by agar disk diffusion and broth microdilution methods. For both strains of staphylococci, farnesol was only able to reverse resistance at a high concentration (150 μM). However, it was very successful at enhancing the antimicrobial efficacy of all of the antibiotics to which the strains were somewhat susceptible. Therefore, synergy testing of farnesol and gentamicin was performed with static biofilms exposed to various concentrations of both agents. Plate counts of harvested biofilm cells at 0, 4, and 24 h posttreatment indicated that the combined effect of gentamicin at 2.5 times the MIC and farnesol at 100 μM (22 μg/ml) was able to reduce bacterial populations by more than 2 log units, demonstrating synergy between the two antimicrobial agents. This observed sensitization of resistant strains to antimicrobials and the observed synergistic effect with gentamicin indicate a potential application for farnesol as an adjuvant therapeutic agent for the prevention of biofilm-related infections and promotion of drug resistance reversal. PMID:16569866

  8. Inhibitory effect of direct electric field and HA-ZnO composites on S. aureus biofilm formation.

    PubMed

    Boda, Sunil Kumar; Bajpai, Indu; Basu, Bikramjit

    2016-08-01

    In addressing the issue of prosthetic infection, we demonstrate herein how direct electric field (DC EF) stimulation can effectively inhibit biofilm formation, when pathogenic Staphylococcus aureus (MRSA, USA 300) are grown on HA-xZnO (x = 0, 5, 7.5, and 10 wt %) biocomposites in vitro. After bacterial preincubation for 4 h, a low intensity DC EF (1V/cm) was applied for different time periods (t = 6, 12, 18, and 24 h). The bacterial viability and biofilm maturation were evaluated by a combination of biochemical assays, fluorescence/confocal microscopy, and flow cytometry. The results confirm a time-dependent and composition-independent decrease in bacterial viability and biofilm formation on HA-xZnO composites w.r.t EF-treated HA. Flow cytometry analysis indicated that 12 h EF application resulted in membrane depolarization of ∼35% of S. aureus populations on HA-xZnO composites. The live/dead assay results revealed ∼60% decline in viable bacterial numbers with a concomitant 3.5-fold increase in the production of reactive oxygen species (ROS) after 18 h of EF. The loss in bacterial viability and biofilm instability is due to the synergistic bactericidal action of ZnO and EF. Taken together, the use of engineered biomaterial substrate with antimicrobial reinforcement coupled with continuous low intensity EF application can be adopted to treat prosthetic implant associated infection. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1064-1075, 2016. PMID:26014126

  9. New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging.

    PubMed

    Boudjemaa, Rym; Briandet, Romain; Revest, Matthieu; Jacqueline, Cédric; Caillon, Jocelyne; Fontaine-Aupart, Marie-Pierre; Steenkeste, Karine

    2016-08-01

    Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associated S. aureus infections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistant S. aureus biofilms. To provide a realistic representation of daptomycin action, we optimized an in vitro model built on the basis of our recently published in vivo mouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest that S. aureus biofilm resistance/tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process. PMID:27297479

  10. Evaluation of ethyl N-(2-phenethyl) carbamate analogues as biofilm inhibitors of methicillin resistant Staphylococcus aureus.

    PubMed

    Stephens, Matthew D; Yodsanit, Nisakorn; Melander, Christian

    2016-07-12

    A small molecule library consisting of 45 compounds was synthesized based on the bacterial metabolite ethyl N-(2-phenethyl) carbamate. Screening of the compounds revealed a potent analogue capabale of inhibiting several strains of Methicillin Resistant S. aureus biofilms with low to moderate micromolar IC50 values. PMID:27341658

  11. Staphylococcus aureus isolated from handmade sweets: Biofilm formation, enterotoxigenicity and antimicrobial resistance.

    PubMed

    Kroning, Isabela Schneid; Iglesias, Mariana Almeida; Sehn, Carla Pohl; Valente Gandra, Tatiane Kuka; Mata, Marcia Magalhães; da Silva, Wladimir Padilha

    2016-09-01

    Staphylococcus aureus is the second most important pathogen involved in foodborne outbreaks in Brazil. Because of their widespread distribution and biofilm forming ability, handmade sweets are easily contaminated with S. aureus. The aim of this study was to isolate and identify coagulase-positive staphylococci (CPS) from handmade sweets produced in Pelotas City/Brazil. The virulence potential was checked by evaluating the presence of the staphylococcal enterotoxin genes, icaA and icaD genes, the biofilm forming potential and antimicrobial resistance of the isolates. It was find just S. aureus among the CPS isolates. All the S. aureus isolates had biofilm forming ability on stainless steel and more than half of them on polystyrene surfaces. The majority of the isolates carried the icaA (66.6%) and icaD (58.4%) genes and some of them had the genes encoding enterotoxins A (33.4%) and B (16.6%). Furthermore, the majority of the isolates (83%) were resistant to at least one of the tested antimicrobials and multidrug resistance was observed in 8.4% of the isolates. The isolates had virulence potential, and half of them were enterotoxigenic. In addition, the ability of all the isolates to produce biofilms highlights the danger posed by these potentially virulent microorganisms persisting in food manufacturing environments. PMID:27217365

  12. Staphylococcus epidermidis Esp Degrades Specific Proteins Associated with Staphylococcus aureus Biofilm Formation and Host-Pathogen Interaction

    PubMed Central

    Iwamoto, Takeo; Takada, Koji; Okuda, Ken-ichi; Tajima, Akiko; Iwase, Tadayuki

    2013-01-01

    Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (EspS235A) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction. PMID:23316041

  13. Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers.

    PubMed

    Kruszewski, Kristen M; Nistico, Laura; Longwell, Mark J; Hynes, Matthew J; Maurer, Joshua A; Hall-Stoodley, Luanne; Gawalt, Ellen S

    2013-05-01

    Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (-CH3) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an "active" antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively. PMID:23498233

  14. Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

    PubMed Central

    Manner, Suvi; Skogman, Malena; Goeres, Darla; Vuorela, Pia; Fallarero, Adyary

    2013-01-01

    When single-cell (or suspended) bacteria switch into the biofilm lifestyle, they become less susceptible to antimicrobials, imposing the need for anti-biofilms research. Flavonoids are among the most extensively studied natural compounds with an unprecedented amount of bioactivity claims. Most studies focus on the antibacterial effects against suspended cells; fewer reports have researched their anti-biofilm properties. Here, a high throughput phenotypic platform was utilized to screen for the inhibitory activity of 500 flavonoids, including natural and synthetic derivatives, against Staphylococcus aureus biofilms. Since discrepancies among results from earlier antibacterial studies on flavonoids had been noted, the current study aimed to minimize sources of variations. After the first screen, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, exclusion criteria combining bioactivity and selectivity identified two synthetic flavans as the most promising. The body of data reported here serves three main purposes. First, it offers an improved methodological workflow for anti-biofilm screens of chemical libraries taking into account the (many times ignored) connections between anti-biofilm and antibacterial properties. This is particularly relevant for the study of flavonoids and other natural products. Second, it provides a large and freely available anti-biofilm bioactivity dataset that expands the knowledge on flavonoids and paves the way for future structure-activity relationship studies and structural optimizations. Finally, it identifies two new flavans that can successfully act on biofilms, as well as on suspended bacteria and represent more feasible antibacterial candidates. PMID:24071942

  15. Impact of growth temperature and surface type on the resistance of Pseudomonas aeruginosa and Staphylococcus aureus biofilms to disinfectants.

    PubMed

    Abdallah, Marwan; Khelissa, Oussama; Ibrahim, Ali; Benoliel, Corinne; Heliot, Laurent; Dhulster, Pascal; Chihib, Nour-Eddine

    2015-12-01

    Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus on food-contact-surfaces represents a significant risk for the public health. In this context, the present study investigates the relationship between the environmental conditions of biofilm formation and the resistance to disinfectants. Therefore, a static biofilm reactor, called NEC-Biofilm System, was established in order to study the effect of growth temperature (20, 30 and 37°C), and of the surface type (stainless steel and polycarbonate), on biofilm resistance to disinfectants. These conditions were selected to mimic the biofilm formation on abiotic surfaces of food processing industries. The antibiofilm assays were performed on biofilms grown during 24 h. The results showed that the growth temperature influenced significantly the biofilm resistance to disinfectants. These data also revealed that the growth temperature has a significant effect on the biofilm structure of both bacteria. Furthermore, the increase of the biofilm growth temperature increased significantly the algD transcript level in sessile P. aeruginosa cells, whereas the icaA one was not affected in S. aureus cells. Overall, our findings show that the biofilm structure and matrix cannot fully explain the biofilm resistance to disinfectant agents. Nevertheless, it underlines the intimate link between environmental conditions, commonly met in food sectors, and the biofilm resistance to disinfectants. PMID:26233298

  16. Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions

    PubMed Central

    2014-01-01

    Background The biofilm forming bacterium Staphylococcus aureus is responsible for maladies ranging from severe skin infection to major diseases such as bacteremia, endocarditis and osteomyelitis. A flow displacement system was used to grow S. aureus biofilms in four physiologically relevant fluid shear rates (50, 100, 500 and 1000 s-1) to identify proteins that are associated with biofilm. Results Global protein expressions from the membrane and cytosolic fractions of S. aureus biofilm cells grown under the above shear rate conditions are reported. Sixteen proteins in the membrane-enriched fraction and eight proteins in the cytosolic fraction showed significantly altered expression (p < 0.05) under increasing fluid shear. These 24 proteins were identified using nano-LC-ESI-MS/MS. They were found to be associated with various metabolic functions such as glycolysis / TCA pathways, protein synthesis and stress tolerance. Increased fluid shear stress did not influence the expression of two important surface binding proteins: fibronectin-binding and collagen-binding proteins. Conclusions The reported data suggest that while the general metabolic function of the sessile bacteria is minimal under high fluid shear stress conditions, they seem to retain the binding capacity to initiate new infections. PMID:24855455

  17. Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs

    PubMed Central

    Cihalova, Kristyna; Chudobova, Dagmar; Michalek, Petr; Moulick, Amitava; Guran, Roman; Kopel, Pavel; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to β-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)—ampicillin, oxacillin and penicillin—and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99% ± 7% for S. aureus and up to 94% ± 4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79% ± 5% and MRSA up to 16% ± 2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs. PMID:26501270

  18. Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus.

    PubMed

    Nan, Li; Yang, Ke; Ren, Guogang

    2015-06-01

    Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu(2+) ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1ppm (2days) to 4.5ppm (7days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu(2+) ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface. PMID:25842145

  19. Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

    PubMed

    Melchior, M B; van Osch, M H J; Lam, T J G M; Vernooij, J C M; Gaastra, W; Fink-Gremmels, J

    2011-12-01

    Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the β-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed

  20. Comparison of Biofilm Formation between Methicillin-Resistant and Methicillin-Susceptible Isolates of Staphylococcus aureus

    PubMed Central

    Ghasemian, Abdolmajid; Najar Peerayeh, Shahin; Bakhshi, Bita; Mirzaee, Mohsen

    2016-01-01

    Background: The aim of this study was to compare the biofilm formation and the prevalence of biofilm-associated genes between the isolates of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus. Methods: In total, 209 S. aureus isolates were collected. The antibiotic susceptibility test was conducted using nine antibiotics according to the guidelines of Clinical and Laboratory Standards Institute. Phenotypic biofilm formation was performed with microtiter plate assay. The polymerase chain reaction was employed to detect icaA, icaD, icaB, icaC, clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, bbp, mecA, and SCCmec types as well as agr group genes with specific primers. Results: Sixty-four (30.62%) isolates were resistant to methicillin, and 54 (83%) MRSA harbored SCCmec III. Furthermore, 122 (58.3%) isolates belonged to agr group I. Twenty-six (36.1%) MRSA and 42 (28.9%) MSSA isolates were strong biofilm producers (no significant difference). The prevalence of icaA, icaD, icaB, and icaC genes in MSSA isolates was 71, 41, 76, and 72%, respectively. The frequency of clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, and bbp in MSSA was 100, 100, 56, 46, 74, 54, 78, 11, and 1%, respectively. However, in MRSA isolates, the frequency was 97, 97, 64, 51, 76, 56, 79, and 12% with no track of bbp, respectively. Conclusion: Statistical difference between MSSA and MRSA regarding biofilm formation and the frequency of all biofilm-encoding genes was not significant. The majority of the S. aureus isolates harbored clfA, clfB, eno, fib, icaA, and icaD genes. PMID:26948126

  1. Bactericidal Effect of a Photoresponsive Carbon Monoxide-Releasing Nonwoven against Staphylococcus aureus Biofilms.

    PubMed

    Klinger-Strobel, Mareike; Gläser, Steve; Makarewicz, Oliwia; Wyrwa, Ralf; Weisser, Jürgen; Pletz, Mathias W; Schiller, Alexander

    2016-07-01

    Staphylococcus aureus is a leading pathogen in skin and skin structure infections, including surgical and traumatic infections that are associated with biofilm formation. Because biofilm formation is accompanied by high phenotypic resistance of the embedded bacteria, they are almost impossible to eradicate by conventional antibiotics. Therefore, alternative therapeutic strategies are of high interest. We generated nanostructured hybrid nonwovens via the electrospinning of a photoresponsive carbon monoxide (CO)-releasing molecule [CORM-1, Mn2(CO)10] and the polymer polylactide. This nonwoven showed a CO-induced antimicrobial activity that was sufficient to reduce the biofilm-embedded bacteria by 70% after photostimulation at 405 nm. The released CO increased the concentration of reactive oxygen species (ROS) in the biofilms, suggesting that in addition to inhibiting the electron transport chain, ROS might play a role in the antimicrobial activity of CORMs on S. aureus The nonwoven showed increased cytotoxicity on eukaryotic cells after longer exposure, most probably due to the released lactic acid, that might be acceptable for local and short-time treatments. Therefore, CO-releasing nonwovens might be a promising local antimicrobial therapy against biofilm-associated skin wound infections. PMID:27114272

  2. Genotypically Different Clones of Staphylococcus aureus Are Diverse in the Antimicrobial Susceptibility Patterns and Biofilm Formations

    PubMed Central

    Atshan, Salman Sahab; Nor Shamsudin, Mariana; Lung, Leslie Than Thian; Sekawi, Zamberi; Pei Pei, Chong; Karunanidhi, Arunkumar; Jeevajothi Nathan, Jayakayatri; Mateg Ali, Alreshidi; Ghaznavi-Rad, Ehsanollah; Abduljaleel, Salwa A.; Awang Hamat, Rukman

    2013-01-01

    This study evaluated whether genotypically different clinical isolates of S. aureus have similar susceptibilities to individual antibiotics. It further aims to check the impact of biofilm on the in vitro activity of vancomycin, daptomycin, linezolid, and tigecycline against S. aureus clones. The study used a total of 60 different clinical MSSA and MRSA isolates. Susceptibilities were performed in planktonic cultures by macrobroth dilution and epsilon-test (E test) system. Biofilm production was determined using an adherent plate assay. The efficacy of antimicrobial activities against biofilms formation was checked using confocal laser scanning microscopy (CLSM). The study found that similar and different spa, MLST, and SCCmec types displayed high variation in their susceptibilities to antibiotics with tigecycline and daptomycin being the most effective. The biofilms were found resistant to high concentrations of most antibiotics tested with daptomycin being the most effective drug used in adhesive biofilms. A considerable difference exists among similar and various clone types against antibiotics tested. This variation could have contributed to the degree of virulence even within the same clonal genotype and enhanced heterogeneity in the infection potential. Thus, the development of a rapid and precise identification profile for each clone in human infections is important. PMID:24455699

  3. Eugenol: A Phyto-Compound Effective against Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Clinical Strain Biofilms

    PubMed Central

    Yadav, Mukesh Kumar; Chae, Sung-Won; Im, Gi Jung; Chung, Jae-Woo; Song, Jae-Jun

    2015-01-01

    Background Inhibition and eradication of Staphylococcus aureus biofilms with conventional antibiotic is difficult, and the treatment is further complicated by the rise of antibiotic resistance among staphylococci. Consequently, there is a need for novel antimicrobials that can treat biofilm-related infections and decrease antibiotics burden. Natural compounds such as eugenol with anti-microbial properties are attractive agents that could reduce the use of conventional antibiotics. In this study we evaluated the effect of eugenol on MRSA and MSSA biofilms in vitro and bacterial colonization in vivo. Methods and Results Effect of eugenol on in vitro biofilm and in vivo colonization were studied using microtiter plate assay and otitis media-rat model respectively. The architecture of in vitro biofilms and in vivo colonization of bacteria was viewed with SEM. Real-time RT-PCR was used to study gene expression. Check board method was used to study the synergistic effects of eugenol and carvacrol on established biofilms. Eugenol significantly inhibited biofilms growth of MRSA and MSSA in vitro in a concentration-dependent manner. Eugenol at MIC or 2×MIC effectively eradicated the pre-established biofilms of MRSA and MSSA clinical strains. In vivo, sub-MIC of eugenol significantly decreased 88% S. aureus colonization in rat middle ear. Eugenol was observed to damage the cell-membrane and cause a leakage of the cell contents. At sub-inhibitory concentration, it decreases the expression of biofilm-and enterotoxin-related genes. Eugenol showed a synergistic effect with carvacrol on the eradication of pre-established biofilms. Conclusion/Major Finding This study demonstrated that eugenol exhibits notable activity against MRSA and MSSA clinical strains biofilms. Eugenol inhibited biofilm formation, disrupted the cell-to-cell connections, detached the existing biofilms, and killed the bacteria in biofilms of both MRSA and MSSA with equal effectiveness. Therefore, eugenol may

  4. Oral administration of the broad-spectrum antibiofilm compound toremifene inhibits Candida albicans and Staphylococcus aureus biofilm formation in vivo.

    PubMed

    De Cremer, Kaat; Delattin, Nicolas; De Brucker, Katrijn; Peeters, Annelies; Kucharíková, Soña; Gerits, Evelien; Verstraeten, Natalie; Michiels, Jan; Van Dijck, Patrick; Cammue, Bruno P A; Thevissen, Karin

    2014-12-01

    We here report on the in vitro activity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, including Candida albicans, Candida glabrata, Candida dubliniensis, Candida krusei, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We validated the in vivo efficacy of orally administered toremifene against C. albicans and S. aureus biofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound. PMID:25288093

  5. Comparison of the In vitro Activity of Five Antimicrobial Drugs against Staphylococcus pseudintermedius and Staphylococcus aureus Biofilms.

    PubMed

    Ferran, Aude A; Liu, JingJing; Toutain, Pierre-Louis; Bousquet-Mélou, Alain

    2016-01-01

    Resistance in canine pathogenic staphylococci is necessitating re-evaluation of the current antimicrobial treatments especially for biofilm-associated infections. Long, repeated treatments are often required to control such infections due to the tolerance of bacteria within the biofilm. To comply with the goal of better antibiotic stewardship in veterinary medicine, the efficacies of the available drugs need to be directly assessed on bacterial biofilms. We compared the activities of amoxicillin, cefalexin, clindamycin, doxycycline, and marbofloxacin on in vitro biofilms of Staphylococcus pseudintermedius and Staphylococcus aureus. Exposure of biofilms for 15 h to maximum concentrations of the antibiotics achievable in canine plasma only reduced biofilm bacteria by 0.5-2.0 log10 CFU, compared to the control, except for marbofloxacin which reduced S. aureus biofilms by 5.4 log10 CFU. Two-antibiotic combinations did not improve, and even decreased, bacterial killing. In comparison, 5 min-exposure to 2% chlorhexidine reduced biofilms of the two tested strains by 4 log10 CFU. Our results showed that S. pseudintermedius and S. aureus biofilms were highly tolerant to all the drugs tested, consistent with the treatment failures observed in practice. Under our in vitro conditions, the use of chlorhexidine was more efficacious than antimicrobials to reduce S. pseudintermedius biofilm. PMID:27531995

  6. Comparison of the In vitro Activity of Five Antimicrobial Drugs against Staphylococcus pseudintermedius and Staphylococcus aureus Biofilms

    PubMed Central

    Ferran, Aude A.; Liu, JingJing; Toutain, Pierre-Louis; Bousquet-Mélou, Alain

    2016-01-01

    Resistance in canine pathogenic staphylococci is necessitating re-evaluation of the current antimicrobial treatments especially for biofilm-associated infections. Long, repeated treatments are often required to control such infections due to the tolerance of bacteria within the biofilm. To comply with the goal of better antibiotic stewardship in veterinary medicine, the efficacies of the available drugs need to be directly assessed on bacterial biofilms. We compared the activities of amoxicillin, cefalexin, clindamycin, doxycycline, and marbofloxacin on in vitro biofilms of Staphylococcus pseudintermedius and Staphylococcus aureus. Exposure of biofilms for 15 h to maximum concentrations of the antibiotics achievable in canine plasma only reduced biofilm bacteria by 0.5–2.0 log10 CFU, compared to the control, except for marbofloxacin which reduced S. aureus biofilms by 5.4 log10 CFU. Two-antibiotic combinations did not improve, and even decreased, bacterial killing. In comparison, 5 min-exposure to 2% chlorhexidine reduced biofilms of the two tested strains by 4 log10 CFU. Our results showed that S. pseudintermedius and S. aureus biofilms were highly tolerant to all the drugs tested, consistent with the treatment failures observed in practice. Under our in vitro conditions, the use of chlorhexidine was more efficacious than antimicrobials to reduce S. pseudintermedius biofilm. PMID:27531995

  7. Characterization of the effect of serum and chelating agents on Staphylococcus aureus biofilm formation; chelating agents augment biofilm formation through clumping factor B

    NASA Astrophysics Data System (ADS)

    Abraham, Nabil Mathew

    Staphylococcus aureus is the causative agent of a diverse array of acute and chronic infections, and some these infections, including infective endocarditis, joint infections, and medical device-associated bloodstream infections, depend upon its capacity to form tenacious biofilms on surfaces. Inserted medical devices such as intravenous catheters, pacemakers, and artificial heart valves save lives, but unfortunately, they can also serve as a substrate on which S. aureus can form a biofilm, attributing S. aureus as a leading cause of medical device-related infections. The major aim of this work was take compounds to which S. aureus would be exposed during infection and to investigate their effects on its capacity to form a biofilm. More specifically, the project investigated the effects of serum, and thereafter of catheter lock solutions on biofilm formation by S. aureus. Pre-coating polystyrene with serum is frequently used as a method to augment biofilm formation. The effect of pre-coating with serum is due to the deposition of extracellular matrix components onto the polystyrene, which are then recognized by MSCRAMMs. We therefore hypothesized that the major component of blood, serum, would induce biofilm formation. Surprisingly, serum actually inhibited biofilm formation. The inhibitory activity was due to a small molecular weight, heat-stable, non-proteinaceous component/s of serum. Serum-mediated inhibition of biofilm formation may represent a previously uncharacterized aspect of host innate immunity that targets the expression of a key bacterial virulence factor: the ability to establish a resistant biofilm. Metal ion chelators like sodium citrate are frequently chosen to lock intravenous catheters because they are regarded as potent inhibitors of bacterial biofilm formation and viability. We found that, while chelating compounds abolished biofilm formation in most strains of S. aureus, they actually augmented the phenotype in a subset of strains. We

  8. Staphylococcus aureus extracellular adherence protein contributes to biofilm formation in the presence of serum

    PubMed Central

    Thompson, Karl M.; Abraham, Nabil; Jefferson, Kimberly K.

    2010-01-01

    Staphylococcus aureus extracellular adherence protein (EAP) is secreted, but it can redock on the bacterial cell surface via neutral phosphatase (Nptase). EAP binds to certain blood proteins and to itself, and through these affinities, it contributes to adherence and aggregation. It has been demonstrated previously that EAP expression is iron regulated and it contributes to biofilm formation under iron-deplete conditions. In this study, we found that EAP and Nptase also play a role in biofilm formation under iron-replete conditions in the presence of human serum. PMID:20199571

  9. Presence of Pseudomonas aeruginosa influences biofilm formation and surface protein expression of Staphylococcus aureus.

    PubMed

    Kumar, Amit; Ting, Yen Peng

    2015-11-01

    Although Staphylococcus aureus and Pseudomonas aeruginosa can individually colonize and infect their hosts, the commensalistic effect of the two is more tenacious and lethal. In this study, it was shown that in co-culture with P. aeruginosa, a sub-population of S. aureus exhibited improved resistance to kanamycin by selection of small colony variant (SCV) phenotype. Additionally, biofilm formation by the two bacteria was denser in the co-culture, compared with biofilm formed in individual pure cultures. Using Atomic Force Microscope (AFM) force spectroscopy for single cells, it was demonstrated that S. aureus cultured in the presence of P. aeruginosa bound more tenaciously to substrates. Surface-shaved peptides were isolated and identified using ultra-performance liquid chromatography-quadrupole-time of flight and a homology search program spider. Results indicated that serine-rich adhesin, extracellular matrix binding protein and other putative adhesion proteins could be responsible for the enhanced attachment of S. aureus in the co-culture. Besides, several other proteins were differentially expressed, indicating the occurrence of a range of other interactions. Of particular interest was a multidrug resistant protein named ABC transporter permease which is known to expel xenobiotics out of the cells. Positive regulation of this protein could be involved in the SCV selection of S. aureus in the co-culture. PMID:25925222

  10. In Vitro Activities of Telavancin and Vancomycin against Biofilm-Producing Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis Strains▿

    PubMed Central

    LaPlante, Kerry L.; Mermel, Leonard A.

    2009-01-01

    We investigated the activities of telavancin and vancomycin against biofilm-producing Staphylococcus and Enterococcus strains. At clinically attainable concentrations, telavancin was active against bacteria embedded in biofilm (minimal biofilm eradication concentration [MBEC], 0.125 to 2 μg/ml) and inhibited biofilm formation at concentrations below the MIC. Vancomycin did not demonstrate the same activity (MBEC, ≥512 μg/ml) against Staphylococcus aureus and Enterococcus faecalis. Telavancin may have a unique role in biofilm-associated infections. PMID:19451302

  11. Effect of licorice extract on cell viability, biofilm formation and exotoxin production by Staphylococcus aureus.

    PubMed

    Rohinishree, Yadahalli Shrihari; Negi, Pradeep Singh

    2016-02-01

    Staphylococcus aureus is one of the most significant clinical pathogen, as it causes infections to humans and animals. Even though several antibiotics and other treatments have been used to control S. aureus infections and intoxication, bacterium is able to adapt, survive and produces exotoxins. Licorice (Glycyrrhiza glabra L.) has been used traditionally in various medicinal (antimicrobial) preparations, and Glycyrrhizic acid (GA) is the major active constituents present in it. In the present investigation the effect of licorice extract on methicillin susceptible S. aureus (FRI 722) and methicillin resistant S. aureus (ATCC 43300) growth and toxin production was studied. The MIC of licorice extract was found to be 0.25 and 2.5 mg GA ml(-1) against S. aureus FRI 722 and S. aureus ATCC 43300, respectively. Inhibition of biofilm formation was observed even at very low concentration (25 μg GA ml(-1)). Gradual decrease in expression and production of exotoxins such as α and β hemolysins and enterotoxin B was observed with the increasing concentrations of licorice extract, however, suboptimal concentration induced the expression of some of the virulence genes. This study indicated efficacy of licorice extract in controlling growth and pathogenicity of both methicillin susceptible and methicillin resistant S. aureus, however, the mechanisms of survival and toxin production at suboptimal concentration needs further study. PMID:27162389

  12. Inhibitory effect of biocides on the viable masses and matrices of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

    PubMed

    Toté, K; Horemans, T; Vanden Berghe, D; Maes, L; Cos, P

    2010-05-01

    Bacteria and matrix are essential for the development of biofilms, and assays should therefore target both components. The current European guidelines for biocidal efficacy testing are not adequate for sessile microorganisms; hence, alternative discriminatory test protocols should be used. The activities of a broad range of biocides on Staphylococcus aureus and Pseudomonas aeruginosa biofilms were evaluated using such in vitro assays. Nearly all selected biocides showed a significant decrease in S. aureus biofilm viability, with sodium hypochlorite and peracetic acid as the most active biocides. Only hydrogen peroxide and sodium hypochlorite showed some inhibitory effect on the matrix. Treatment of P. aeruginosa biofilms was roughly comparable to that of S. aureus biofilms. Peracetic acid was the most active on viable mass within 1 min of contact. Isopropanol ensured a greater than 99.999% reduction of P. aeruginosa viability after at least 30 min of contact. Comparable to results with S. aureus, sodium hypochlorite and hydrogen peroxide markedly reduced the P. aeruginosa matrix. This study clearly demonstrated that despite their aspecific mechanisms of action, most biocides were active only against biofilm bacteria, leaving the matrix undisturbed. Only hydrogen peroxide and sodium hypochlorite were active on both the biofilm matrix and the viable mass, making them the better antibiofilm agents. In addition, this study emphasizes the need for updated and standardized guidelines for biofilm susceptibility testing of biocides. PMID:20363795

  13. Image-based fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms

    NASA Astrophysics Data System (ADS)

    Daddi Oubekka, S.; Briandet, R.; Waharte, F.; Fontaine-Aupart, M.-P.; Steenkeste, K.

    2011-07-01

    The diffusion capabilities of free fluorophores inside the heterogeneous three dimensional structure of Staphylococcus aureus biofilm were studied by an original image-based Fluorescence Recovery After Photobleaching method. The study was extended to BODIPY-vancomycin in order to better understand the mechanisms involved in the high tolerance of the bacteria embedded in a biofilm to the antibiotic.

  14. Antimicrobial Activity of Selected Phytochemicals against Escherichia coli and Staphylococcus aureus and Their Biofilms.

    PubMed

    Monte, Joana; Abreu, Ana C; Borges, Anabela; Simões, Lúcia Chaves; Simões, Manuel

    2014-01-01

    Bacteria can be resistant to multiple antibiotics and we are fast approaching a time when antibiotics will not work on some bacterial infections. New antimicrobial compounds are urgently necessary. Plants are considered the greatest source to obtain new antimicrobials. This study aimed to assess the antimicrobial activity of four phytochemicals-7-hydroxycoumarin (7-HC), indole-3-carbinol (I3C), salicylic acid (SA) and saponin (SP)-against Escherichia coli and Staphylococcus aureus, either as planktonic cells or as biofilms. These bacteria are commonly found in hospital-acquired infections. Some aspects on the phytochemicals mode of action, including surface charge, hydrophobicity, motility and quorum-sensing inhibition (QSI) were investigated. In addition, the phytochemicals were combined with three antibiotics in order to assess any synergistic effect. 7-HC and I3C were the most effective phytochemicals against E. coli and S. aureus. Both phytochemicals affected the motility and quorum-sensing (QS) activity, which means that they can play an important role in the interference of cell-cell interactions and in biofilm formation and control. However, total biofilm removal was not achieved with any of the selected phytochemicals. Dual combinations between tetracycline (TET), erythromycin (ERY) and ciprofloxacin (CIP) and I3C produced synergistic effects against S. aureus resistant strains. The overall results demonstrates the potential of phytochemicals to control the growth of E. coli and S. aureus in both planktonic and biofilm states. In addition, the phytochemicals demonstrated the potential to act synergistically with antibiotics, contributing to the recycling of old antibiotics that were once considered ineffective due to resistance problems. PMID:25437810

  15. Antimicrobial Activity of Selected Phytochemicals against Escherichia coli and Staphylococcus aureus and Their Biofilms

    PubMed Central

    Monte, Joana; Abreu, Ana C.; Borges, Anabela; Simões, Lúcia Chaves; Simões, Manuel

    2014-01-01

    Abstract Bacteria can be resistant to multiple antibiotics and we are fast approaching a time when antibiotics will not work on some bacterial infections. New antimicrobial compounds are urgently necessary. Plants are considered the greatest source to obtain new antimicrobials. This study aimed to assess the antimicrobial activity of four phytochemicals—7-hydroxycoumarin (7-HC), indole-3-carbinol (I3C), salicylic acid (SA) and saponin (SP)—against Escherichia coli and Staphylococcus aureus, either as planktonic cells or as biofilms. These bacteria are commonly found in hospital-acquired infections. Some aspects on the phytochemicals mode of action, including surface charge, hydrophobicity, motility and quorum-sensing inhibition (QSI) were investigated. In addition, the phytochemicals were combined with three antibiotics in order to assess any synergistic effect. 7-HC and I3C were the most effective phytochemicals against E. coli and S. aureus. Both phytochemicals affected the motility and quorum-sensing (QS) activity, which means that they can play an important role in the interference of cell-cell interactions and in biofilm formation and control. However, total biofilm removal was not achieved with any of the selected phytochemicals. Dual combinations between tetracycline (TET), erythromycin (ERY) and ciprofloxacin (CIP) and I3C produced synergistic effects against S. aureus resistant strains. The overall results demonstrates the potential of phytochemicals to control the growth of E. coli and S. aureus in both planktonic and biofilm states. In addition, the phytochemicals demonstrated the potential to act synergistically with antibiotics, contributing to the recycling of old antibiotics that were once considered ineffective due to resistance problems. PMID:25437810

  16. Regulatory Mutations Impacting Antibiotic Susceptibility in an Established Staphylococcus aureus Biofilm.

    PubMed

    Atwood, Danielle N; Beenken, Karen E; Lantz, Tamara L; Meeker, Daniel G; Lynn, William B; Mills, Weston B; Spencer, Horace J; Smeltzer, Mark S

    2016-03-01

    We previously determined the extent to which mutations of different Staphylococcus aureus regulatory loci impact biofilm formation as assessed under in vitro conditions. Here we extend these studies to determine the extent to which those regulatory loci that had the greatest effect on biofilm formation also impact antibiotic susceptibility. The experiments were done under in vitro and in vivo conditions using two clinical isolates of S. aureus (LAC and UAMS-1) and two functionally diverse antibiotics (daptomycin and ceftaroline). Mutation of the staphylococcal accessory regulator (sarA) or sigB was found to significantly increase susceptibilities to both antibiotics and in both strains in a manner that could not be explained by changes in the MICs. The impact of a mutation in sarA was comparable to that of a mutation in sigB and greater than the impact observed with any other mutant. These results suggest that therapeutic strategies targeting sarA and/or sigB have the greatest potential to facilitate the ability to overcome the intrinsic antibiotic resistance that defines S. aureus biofilm-associated infections. PMID:26824954

  17. Regulatory Mutations Impacting Antibiotic Susceptibility in an Established Staphylococcus aureus Biofilm

    PubMed Central

    Beenken, Karen E.; Lantz, Tamara L.; Meeker, Daniel G.; Lynn, William B.; Mills, Weston B.; Spencer, Horace J.

    2016-01-01

    We previously determined the extent to which mutations of different Staphylococcus aureus regulatory loci impact biofilm formation as assessed under in vitro conditions. Here we extend these studies to determine the extent to which those regulatory loci that had the greatest effect on biofilm formation also impact antibiotic susceptibility. The experiments were done under in vitro and in vivo conditions using two clinical isolates of S. aureus (LAC and UAMS-1) and two functionally diverse antibiotics (daptomycin and ceftaroline). Mutation of the staphylococcal accessory regulator (sarA) or sigB was found to significantly increase susceptibilities to both antibiotics and in both strains in a manner that could not be explained by changes in the MICs. The impact of a mutation in sarA was comparable to that of a mutation in sigB and greater than the impact observed with any other mutant. These results suggest that therapeutic strategies targeting sarA and/or sigB have the greatest potential to facilitate the ability to overcome the intrinsic antibiotic resistance that defines S. aureus biofilm-associated infections. PMID:26824954

  18. Chelating agents exert distinct effects on biofilm formation in Staphylococcus aureus depending on strain background: role for clumping factor B

    PubMed Central

    Abraham, Nabil M.; Lamlertthon, Supaporn; Fowler, Vance G.

    2012-01-01

    Staphylococcus aureus is a leading cause of catheter infections, and biofilm formation plays a key role in the pathogenesis. Metal ion chelators inhibit bacterial biofilm formation and viability, making them attractive candidates as components in catheter lock solutions. The goal of this study was to characterize further the effect of chelators on biofilm formation. The effect of the calcium chelators ethylene glycol tetraacetic acid (EGTA) and trisodium citrate (TSC) on biofilm formation by 30 S. aureus strains was tested. The response to subinhibitory doses of EGTA and TSC varied dramatically depending on strain variation. In some strains, the chelators prevented biofilm formation, in others they had no effect, and they actually enhanced biofilm formation in others. The molecular basis for this phenotypic variability was investigated using two related strains: Newman, in which biofilm formation was inhibited by chelators, and 10833, which formed strong biofilms in the presence of chelators. It was found that deletion of the gene encoding the surface adhesin clumping factor B (clfB) completely eliminated chelator-induced biofilm formation in strain 10833. The role of ClfB in biofilm formation activity in chelators was confirmed in additional strains. It was concluded that biofilm-forming ability varies strikingly depending on strain background, and that ClfB is involved in biofilm formation in the presence EGTA and citrate. These results suggest that subinhibitory doses of chelating agents in catheter lock solutions may actually augment biofilm formation in certain strains of S. aureus, and emphasize the importance of using these agents appropriately so that inhibitory doses are achieved consistently. PMID:22516131

  19. Pharmacokinetic/Pharmacodynamic Correlation of Cefquinome Against Experimental Catheter-Associated Biofilm Infection Due to Staphylococcus aureus

    PubMed Central

    Zhou, Yu-Feng; Shi, Wei; Yu, Yang; Tao, Meng-Ting; Xiong, Yan Q.; Sun, Jian; Liu, Ya-Hong

    2016-01-01

    Biofilm formations play an important role in Staphylococcus aureus pathogenesis and contribute to antibiotic treatment failures in biofilm-associated infections. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profiles of cefquinome against an experimental catheter-related biofilm model due to S. aureus, including three clinical isolates and one non-clinical isolate. The minimal inhibitory concentration (MIC), minimal biofilm inhibitory concentration (MBIC), biofilm bactericidal concentration (BBC), minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) and in vitro time-kill curves of cefquinome were studied in both planktonic and biofilm cells of study S. aureus strains. The in vivo post-antibiotic effects (PAEs), PK profiles and efficacy of cefquinome were performed in the catheter-related biofilm infection model in murine. A sigmoid Emax model was utilized to determine the PK/PD index that best described the dose-response profiles in the model. The MICs and MBICs of cefquinome for the four S. aureus strains were 0.5 and 16 μg/mL, respectively. The BBCs (32–64 μg/mL) and MBECs (64–256 μg/mL) of these study strains were much higher than their corresponding BPC values (1–2 μg/mL). Cefquinome showed time-dependent killing both on planktonic and biofilm cells, but produced much shorter PAEs in biofilm infections. The best-correlated PK/PD parameters of cefquinome for planktonic and biofilm cells were the duration of time that the free drug level exceeded the MIC (fT > MIC, R2 = 96.2%) and the MBIC (fT > MBIC, R2 = 94.7%), respectively. In addition, the AUC24h/MBIC of cefquinome also significantly correlated with the anti-biofilm outcome in this model (R2 = 93.1%). The values of AUC24h/MBIC for biofilm-static and 1-log10-unit biofilm-cidal activity were 22.8 and 35.6 h; respectively. These results indicate that the PK/PD profiles of cefquinome could be used as valuable guidance for

  20. Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and the hemolytic activity of Staphylococcus aureus

    PubMed Central

    Lee, Jin-Hyung; Kim, Yong-Guy; Yong Ryu, Shi; Lee, Jintae

    2016-01-01

    Staphylococcal biofilms are problematic and play a critical role in the persistence of chronic infections because of their abilities to tolerate antimicrobial agents. Thus, the inhibitions of biofilm formation and/or toxin production are viewed as alternative means of controlling Staphylococcus aureus infections. Here, the antibiofilm activities of 560 purified phytochemicals were examined. Alizarin at 10 μg/ml was found to efficiently inhibit biofilm formation by three S. aureus strains and a Staphylococcus epidermidis strain. In addition, two other anthraquinones purpurin and quinalizarin were found to have antibiofilm activity. Binding of Ca2+ by alizarin decreased S. aureus biofilm formation and a calcium-specific chelating agent suppressed the effect of calcium. These three anthraquinones also markedly inhibited the hemolytic activity of S. aureus, and in-line with their antibiofilm activities, increased cell aggregation. A chemical structure-activity relationship study revealed that two hydroxyl units at the C-1 and C-2 positions of anthraquinone play important roles in antibiofilm and anti-hemolytic activities. Transcriptional analyses showed that alizarin repressed the α-hemolysin hla gene, biofilm-related genes (psmα, rbf, and spa), and modulated the expressions of cid/lrg genes (the holin/antiholin system). These findings suggest anthraquinones, especially alizarin, are potentially useful for controlling biofilm formation and the virulence of S. aureus. PMID:26763935

  1. Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and the hemolytic activity of Staphylococcus aureus.

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Yong Ryu, Shi; Lee, Jintae

    2016-01-01

    Staphylococcal biofilms are problematic and play a critical role in the persistence of chronic infections because of their abilities to tolerate antimicrobial agents. Thus, the inhibitions of biofilm formation and/or toxin production are viewed as alternative means of controlling Staphylococcus aureus infections. Here, the antibiofilm activities of 560 purified phytochemicals were examined. Alizarin at 10 μg/ml was found to efficiently inhibit biofilm formation by three S. aureus strains and a Staphylococcus epidermidis strain. In addition, two other anthraquinones purpurin and quinalizarin were found to have antibiofilm activity. Binding of Ca(2+) by alizarin decreased S. aureus biofilm formation and a calcium-specific chelating agent suppressed the effect of calcium. These three anthraquinones also markedly inhibited the hemolytic activity of S. aureus, and in-line with their antibiofilm activities, increased cell aggregation. A chemical structure-activity relationship study revealed that two hydroxyl units at the C-1 and C-2 positions of anthraquinone play important roles in antibiofilm and anti-hemolytic activities. Transcriptional analyses showed that alizarin repressed the α-hemolysin hla gene, biofilm-related genes (psmα, rbf, and spa), and modulated the expressions of cid/lrg genes (the holin/antiholin system). These findings suggest anthraquinones, especially alizarin, are potentially useful for controlling biofilm formation and the virulence of S. aureus. PMID:26763935

  2. Role of Phenol-Soluble Modulins in Formation of Staphylococcus aureus Biofilms in Synovial Fluid

    PubMed Central

    Dastgheyb, Sana S.; Villaruz, Amer E.; Le, Katherine Y.; Tan, Vee Y.; Duong, Anthony C.; Chatterjee, Som S.; Cheung, Gordon Y. C.; Joo, Hwang-Soo; Hickok, Noreen J.

    2015-01-01

    Staphylococcus aureus is a leading cause of prosthetic joint infections, which, as we recently showed, proceed with the involvement of biofilm-like clusters that cause recalcitrance to antibiotic treatment. Here we analyzed why these clusters grow extraordinarily large, reaching macroscopically visible extensions (>1 mm). We found that while specific S. aureus surface proteins are a prerequisite for agglomeration in synovial fluid, low activity of the Agr regulatory system and subsequent low production of the phenol-soluble modulin (PSM) surfactant peptides cause agglomerates to grow to exceptional dimensions. Our results indicate that PSMs function by disrupting interactions of biofilm matrix molecules, such as the polysaccharide intercellular adhesin (PIA), with the bacterial cell surface. Together, our findings support a two-step model of staphylococcal prosthetic joint infection: As we previously reported, interaction of S. aureus surface proteins with host matrix proteins such as fibrin initiates agglomeration; our present results show that, thereafter, the bacterial agglomerates grow to extremely large sizes owing to the lack of PSM expression under the specific conditions present in joints. Our findings provide a mechanistic explanation for the reported extreme resistance of joint infection to antibiotic treatment, lend support to the notions that Agr functionality and PSM production play a major role in defining different forms of S. aureus infection, and have important implications for antistaphylococcal therapeutic strategies. PMID:25964472

  3. Inhibitory activity of thymol on native and mature Gardnerella vaginalis biofilms: in vitro study.

    PubMed

    Braga, Pier Carlo; Dal Sasso, Monica; Culici, Maria; Spallino, Alessandra

    2010-01-01

    Bacterial vaginosis (BV) is the most frequent diagnosis made in women with lower genital tract symptoms. It has recently been observed that 90 % of subjects with BV show the growth of bacteria in the form of biofilms as against only 10% without BV, and that Gardnerella vaginalis was the predominant species. The propensity of G. vaginalis to form biofilm is clinically relevant because this form of growth allows it to tolerate higher concentrations of certain antibiotics, thus increasing the possibilty of recurrent BV even after apparently curative therapy. The aim of this study was to investigate whether thymol (CAS 89-83-8), a molecule present in thyme essential oil, that is credited with having a series of pharmacological properties including antimicrobial and antifungal effects, can interfere with newly formed and mature G. vaginalis biofilms. The ability of G. vaginalis ATCC 49145 and two G. vaginalis strains isolated from human BV to form biofilm in flat-bottomed 96-well microtitre plates was verified, and the effects of thymol concentrations ranging from 1 to 1/16 MIC (minimum inhibitory concentration) on preformed and mature biofilms was investigated by means of spectrophotometric analysis, Nomarski interference contrast microscopy, and fluorescence microscopy with live-dead cell visualisation (SYTO 9 and propidium iodide). Native biofilm was inhibited by concentrations ranging from 1 MIC to 1/8 MIC (32.77% +/- 2.37 to 11.39% +/- 1.46), and mature biofilm was inhibited by concentrations ranging from 1 MIC to 1/4 MIC (26.18% +/- 1.36 to 13.20% +/- 1.44). Nomarski interference contrast and fluorescence microscopy visually confirmed these findings. As biofilm is a multi-factorial phenomenon, the multiple mechanisms of thymol may act on different steps in the evolution of mature biofilm. PMID:21175040

  4. Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes.

    PubMed

    Saá Ibusquiza, P; Herrera, J J R; Cabo, M L

    2011-05-01

    Increase of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 °C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD(90)), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex "cloud-type" structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified. PMID:21356446

  5. Identification of Staphylococcus aureus Proteins Recognized by the Antibody-Mediated Immune Response to a Biofilm Infection

    PubMed Central

    Brady, Rebecca A.; Leid, Jeff G.; Camper, Anne K.; Costerton, J. William; Shirtliff, Mark E.

    2006-01-01

    Staphylococcus aureus causes persistent, recurrent infections (e.g., osteomyelitis) by forming biofilms. To survey the antibody-mediated immune response and identify those proteins that are immunogenic in an S. aureus biofilm infection, the tibias of rabbits were infected with methicillin-resistant S. aureus to produce chronic osteomyelitis. Sera were collected prior to infection and at 14, 28, and 42 days postinfection. The sera were used to perform Western blot assays on total protein from biofilm grown in vitro and separated by two-dimensional gel electrophoresis. Those proteins recognized by host antibodies in the harvested sera were identified via matrix-assisted laser desorption ionization-time of flight analysis. Using protein from mechanically disrupted total and fractionated biofilm protein samples, we identified 26 and 22 immunogens, respectively. These included a cell surface-associated β-lactamase, lipoprotein, lipase, autolysin, and an ABC transporter lipoprotein. Studies were also performed using microarray analyses and confirmed the biofilm-specific up-regulation of most of these genes. Therefore, although the biofilm antigens are recognized by the immune system, the biofilm infection can persist. However, these proteins, when delivered as vaccines, may be important in directing the immune system toward an early and effective antibody-mediated response to prevent chronic S. aureus infections. Previous works have identified S. aureus proteins that are immunogenic during acute infections, such as sepsis. However, this is the first work to identify these immunogens during chronic S. aureus biofilm infections and to simultaneously show the global relationship between the antigens expressed during an in vivo infection and the corresponding in vitro transcriptomic and proteomic gene expression levels. PMID:16714572

  6. Role of Daptomycin in the Induction and Persistence of the Viable but Non-Culturable State of Staphylococcus Aureus Biofilms

    PubMed Central

    Pasquaroli, Sonia; Citterio, Barbara; Di Cesare, Andrea; Amiri, Mehdi; Manti, Anita; Vuotto, Claudia; Biavasco, Francesca

    2014-01-01

    We have recently demonstrated that antibiotic pressure can induce the viable but non-culturable (VBNC) state in Staphylococcus aureus biofilms. Since dormant bacterial cells can undermine anti-infective therapy, a greater understanding of the role of antibiotics of last resort, including daptomycin, is crucial. Methicillin-resistant S. aureus 10850 biofilms were maintained on non-nutrient (NN) agar in the presence or absence of the MIC of daptomycin until loss of culturability. Viable cells were monitored by epifluorescence microscopy and flow cytometry for 150 days. All biofilms reached non-culturability at 40 days and showed a similar amount of viable cells; however, in biofilms exposed to daptomycin, their number remained unchanged throughout the experiment, whereas in those maintained on NN agar alone, no viable cells were detected after 150 days. Gene expression assays showed that after achievement of non-culturability, 16S rDNA and mecA were expressed by all biofilms, whereas glt expression was found only in daptomycin-exposed biofilms. Our findings suggest that low daptomycin concentrations, such as those that are likely to obtain within biofilms, can influence the viability and gene expression of non-culturable S. aureus cells. Resuscitation experiments are needed to establish the VBNC state of daptomycin-exposed biofilms. PMID:25438023

  7. Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms

    PubMed Central

    Stoodley, Paul; Wilson, Suzanne; Hall-Stoodley, Luanne; Boyle, John D.; Lappin-Scott, Hilary M.; Costerton, J. W.

    2001-01-01

    Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 μm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment. PMID:11722913

  8. Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans.

    PubMed

    Verma-Gaur, Jiyoti; Qu, Yue; Harrison, Paul F; Lo, Tricia L; Quenault, Tara; Dagley, Michael J; Bellousoff, Matthew; Powell, David R; Beilharz, Traude H; Traven, Ana

    2015-10-01

    The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease. PMID:26474309

  9. Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans

    PubMed Central

    Harrison, Paul F.; Lo, Tricia L.; Quenault, Tara; Dagley, Michael J.; Bellousoff, Matthew; Powell, David R.; Beilharz, Traude H.; Traven, Ana

    2015-01-01

    The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease. PMID:26474309

  10. Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants.

    PubMed

    Phillips, Priscilla L; Yang, Qingping; Davis, Stephen; Sampson, Edith M; Azeke, John I; Hamad, Afifa; Schultz, Gregory S

    2015-08-01

    An ex vivo porcine skin explant biofilm model that preserves key properties of biofilm attached to skin at different levels of maturity (0-3 days) was used to assess the efficacy of commercially available antimicrobial dressings and topical treatments. Assays were also performed on the subpopulation of antibiotic tolerant biofilm generated by 24 hours of pre-treatment with gentamicin (120× minimal inhibitory concentration) prior to agent exposure. Five types of antimicrobial agents (iodine, silver, polyhexamethylene biguanide, honey and ethanol) and four types of moisture dressings (cotton gauze, sodium carboxymethylcellulose fibre, calcium alginate fibre and cadexomer beads) were assessed. Time-release silver gel and cadexomer iodine dressings were the most effective in reducing mature biofilm [between 5 and 7 logarithmic (log) of 7-log total], whereas all other dressing formulations reduced biofilm between 0·3 and 2 log in 24 or 72 hours with a single exposure. Similar results were found after 24-hour exposure to silver release dressings using an in vivo pig burn wound model, demonstrating correlation between the ex vivo and in vivo models. Results of this study indicate that commonly used microbicidal wound dressings vary widely in their ability to kill mature biofilm and the efficacy is influenced by time of exposure, number of applications, moisture level and agent formulation (sustained release). PMID:24028432

  11. Effect of peracetic acid on biofilms formed by Staphylococcus aureus and Listeria monocytogenes isolated from dairy plants.

    PubMed

    Lee, S H I; Cappato, L P; Corassin, C H; Cruz, A G; Oliveira, C A F

    2016-03-01

    This research investigated the removal of adherent cells of 4 strains of Staphylococcus aureus and 1 Listeria monocytogenes strain (previously isolated from dairy plants) from polystyrene microtiter plates using peracetic acid (PAA, 0.5%) for 15, 30, 60, and 120 s, and the inactivation of biofilms formed by those strains on stainless steel coupons using the same treatment times. In the microtiter plates, PAA removed all S. aureus at 15 s compared with control (no PAA treatment). However, L. monocytogenes biofilm was not affected by any PAA treatment. On the stainless steel surface, epifluorescence microscopy using LIVE/DEAD staining (BacLight, Molecular Probes/Thermo Fisher Scientific, Eugene, OR) showed that all strains were damaged within 15 s, with almost 100% of cells inactivated after 30 s. Results of this trial indicate that, although PAA was able to inactivate both S. aureus and L. monocytogenes monospecies biofilms on stainless steel, it was only able to remove adherent cells of S. aureus from polystyrene microplates. The correct use of PAA is critical for eliminating biofilms formed by S. aureus strains found in dairy plants, although further studies are necessary to determine the optimal PAA treatment for removing biofilms of L. monocytogenes. PMID:26723125

  12. Effects of antimicrobial peptides on Staphylococcus aureus growth and biofilm formation in vitro following isolation from implant-associated infections

    PubMed Central

    Zhao, Guangfeng; Zhong, Huiming; Zhang, Mao; Hong, Yucai

    2015-01-01

    To prevent biomaterial-associated infections, antibiotic agents are recommended for various medical conditions requiring biomaterial implants, but resistance often appears after the introduction of antibiotics into clinical use. Therefore, new strategies for the prevention or treatment for biomaterial-associated infections are required. The purpose of this study was to evaluate the effects of antimicrobial peptides on growth and biofilm formation of Staphylococcus aureus isolated from implant-associated infections. A total of 20 patients with culture-proven staphylococcal infection associated with stable orthopedic implants were selected as the experimental group. S. aureus were isolated from tissue biopsies for identification, the isolated strains were mixed with Tet213 incubated at 37°C and viable bactrial number of S. aureus was counted. For the biofilm formation, the broad spectrum AMP Tet213 was selected and loaded onto the Ti coating first. At the same time Ti coated with Tet213 were mixed with S. aureus in vitro to form biofilm. After 30 min, 2 h, 4 h, 6 h, 8 h, the population of S. aureus in the biofilm was counted. Tet213 showed significant antibacterial effect on 16 strains (P < 0.05, Table 1). The inhibition rate reached above 80% among 12 strains of the clinically isolated strain. In biofilm experiments, counts of the NO. 1, 2, 3, 4 strains in biofilms decreased significantly after 2 h (P < 0.05), while there was no obvious difference in counts of NO. 5 strain (P > 0.05). The broad spectrum AMP Tet213 could strongly reduce the growth and biofilm formation of S. aureus in vitro, and the use of this might be an important new approach to target implant-associated infections. PMID:25785171

  13. Bap, a Biofilm Matrix Protein of Staphylococcus aureus Prevents Cellular Internalization through Binding to GP96 Host Receptor

    PubMed Central

    Valle, Jaione; Latasa, Cristina; Gil, Carmen; Toledo-Arana, Alejandro; Solano, Cristina; Penadés, José R.; Lasa, Iñigo

    2012-01-01

    The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilm-associated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections. PMID:22876182

  14. Association between Methicillin Susceptibility and Biofilm Regulation in Staphylococcus aureus Isolates from Device-Related Infections▿ †

    PubMed Central

    O'Neill, Eoghan; Pozzi, Clarissa; Houston, Patrick; Smyth, Davida; Humphreys, Hilary; Robinson, D. Ashley; O'Gara, James P.

    2007-01-01

    Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucose-induced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillin-sensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolate's genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development. PMID:17329452

  15. Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones of Staphylococcus aureus

    PubMed Central

    Atshan, Salman Sahab; Nor Shamsudin, Mariana; Sekawi, Zamberi; Lung, Leslie Than Thian; Hamat, Rukman Awang; Karunanidhi, Arunkumar; Mateg Ali, Alreshidi; Ghaznavi-Rad, Ehsanollah; Ghasemzadeh-Moghaddam, Hamed; Chong Seng, Johnson Shueh; Nathan, Jayakayatri Jeevajothi; Pei Pei, Chong

    2012-01-01

    Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the same spa type were found to have similar properties in adheringto the polystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes). icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC) was confirmed by RT-PCR. PMID:22701309

  16. Comparison of the Antibiotic Activities of Daptomycin, Vancomycin, and the Investigational Fluoroquinolone Delafloxacin against Biofilms from Staphylococcus aureus Clinical Isolates

    PubMed Central

    Siala, Wafi; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M.; Hallin, Marie; Denis, Olivier

    2014-01-01

    Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726–2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local

  17. Biofilm production among methicillin resistant Staphylococcus aureus strains isolated from catheterized patients with urinary tract infection.

    PubMed

    Rahimi, Fateh; Katouli, Mohammad; Karimi, Sharmin

    2016-09-01

    Between June 2011 and May 2014, we isolated a total of 419 Staphylococcus aureus strains from catheterized patients with UTI in a referral hospital in Tehran. Of these, 108 were identified as methicillin resistant (MRSA) based on their phenotypic resistance to oxacillin and the presence of mecA gene. The MRSA isolates were tested for their clonality using a combination of PFGE, prophage typing, SCCmec and ccr typing and examined for their biofilm formation as well as their resistance against 17 antibiotics. In all, 15 common pulsotypes consisted of 105 isolates and 3 single types were identified among the MRSA strains of which, 97% carried SCCmec type III and type 3 ccr. Eighty three (77%) strains were positive for biofilm formation and also carried icaA and icaD genes. Moreover, agr group III and its related tst gene were detected in 81% and 77% of biofilm producing strains, respectively 105 of the 108 MRSA were multidrug resistant with 82.4% being resistant to more than 10 antibiotics. Strains with SCCmec type IV and type 2 ccr, contained SGA and SGL prophage types, were positive for pvl gene and belonged to single PFGE types. This study highlights the important role of biofilm formation and virulence factors of MRSA strains in catheterized patients. PMID:27374894

  18. Coral-Associated Bacteria as a Promising Antibiofilm Agent against Methicillin-Resistant and -Susceptible Staphylococcus aureus Biofilms

    PubMed Central

    Gowrishankar, Shanmugaraj; Duncun Mosioma, Nyagwencha; Karutha Pandian, Shunmugiah

    2012-01-01

    The current study deals with the evaluation of two coral-associated bacterial (CAB) extracts to inhibit the biofilm synthesis in vitro as well as the virulence production like hemolysin and exopolysaccharide (EPS), and also to assess their ability to modify the adhesion properties, that is cell surface hydrophobicity (CSH) of methicillin-resistant (MRSA) and -susceptible Staphylococcus aureus (MSSA). Out of nine CAB screened, the ethyl acetate extract of CAB-E2 (Bacillus firmus) and CAB-E4 (Vibrio parahemolyticus) have shown excellent antibiofilm activity against S. aureus. CAB-E2 reduced the production of EPS (57–79%) and hemolysin (43–70%), which ultimately resulted in the significant inhibition of biofilms (80–87%) formed by both MRSA and MSSA. Similarly, CAB-E4 was also found to decrease the production of EPS (43–57%), hemolysin (43–57%) and biofilms (80–85%) of test pathogens. CLSM analysis also proved the antibiofilm efficacy of CAB extracts. Furthermore, the CAB extracts strongly decreased the CSH of S. aureus. Additionally, FT-IR analysis of S. aureus treated with CAB extracts evidenced the reduction in cellular components compared to their respective controls. Thus, the present study reports for the first time, B. firmus—a coral-associated bacterium, as a promising source of antibiofilm agent against the recalcitrant biofilms formed by multidrug resistant S. aureus. PMID:22988476

  19. Exopolysaccharide Biosynthesis Enables Mature Biofilm Formation on Abiotic Surfaces by Herbaspirillum seropedicae

    PubMed Central

    Balsanelli, Eduardo; de Baura, Válter Antonio; Pedrosa, Fábio de Oliveira; de Souza, Emanuel Maltempi; Monteiro, Rose Adele

    2014-01-01

    H. seropedicae associates endophytically and epiphytically with important poaceous crops and is capable of promoting their growth. The molecular mechanisms involved in plant colonization by this microrganism are not fully understood. Exopolysaccharides (EPS) are usually necessary for bacterial attachment to solid surfaces, to other bacteria, and to form biofilms. The role of H. seropedicae SmR1 exopolysaccharide in biofilm formation on both inert and plant substrates was assessed by characterization of a mutant in the espB gene which codes for a glucosyltransferase. The mutant strain was severely affected in EPS production and biofilm formation on glass wool. In contrast, the plant colonization capacity of the mutant strain was not altered when compared to the parental strain. The requirement of EPS for biofilm formation on inert surface was reinforced by the induction of eps genes in biofilms grown on glass and polypropylene. On the other hand, a strong repression of eps genes was observed in H. seropedicae cells adhered to maize roots. Our data suggest that H. seropedicae EPS is a structural component of mature biofilms, but this development stage of biofilm is not achieved during plant colonization. PMID:25310013

  20. Antimicrobial activity of zinc and titanium dioxide nanoparticles against biofilm-producing methicillin-resistant Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Jesline, A.; John, Neetu P.; Narayanan, P. M.; Vani, C.; Murugan, Sevanan

    2015-02-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens responsible for a wide spectrum of infections and the emergence of bacterial resistance to antibiotics has lead to treatment drawbacks towards large number of drugs. Formation of biofilms is the main contributing factor to antibiotic resistance. The development of reliable processes for the synthesis of zinc oxide nanoparticles is an important aspect of nanotechnology today. Zinc oxide and titanium dioxide nanoparticles comprise well-known inhibitory and bactericidal effects. Emergence of antimicrobial resistance by pathogenic bacteria is a major health problem in recent years. This study was designed to determine the efficacy of zinc and titanium dioxide nanoparticles against biofilm producing methicillin-resistant S. aureus. Biofilm production was detected by tissue culture plate method. Out of 30 MRSA isolates, 22 isolates showed strong biofilm production and 2 showed weak and moderate biofilm formation. Two strong and weak biofilm-producing methicillin-resistant S. aureus isolates were subjected to antimicrobial activity using commercially available zinc and titanium dioxide nanoparticles. Thus, the nanoparticles showed considerably good activity against the isolates, and it can be concluded that they may act as promising, antibacterial agents in the coming years.

  1. Antimicrobial efficacy of combined clarithromycin plus daptomycin against biofilms-formed methicillin-resistant Staphylococcus aureus on titanium medical devices.

    PubMed

    Fujimura, Shigeru; Sato, Tetsuro; Hayakawa, Sachiko; Kawamura, Masato; Furukawa, Emiko; Watanabe, Akira

    2015-10-01

    In vitro efficacy of combined eradication therapy with clarithromycin and daptomycin against biofilm-formed methicillin-resistant Staphylococcus aureus on the orthopedic titanium devices was evaluated. The bactericidal effect of this antibiotic was investigated by a re-culture test, the scanning electron microscopy, and fluorescence microscopy using a double-staining dyes. Clarithromycin decreased the amount to half in 24 h. Although MRSA biofilms were not eradicated with clarithromycin or daptomycin alone, clarithromycin combined with daptomycin was useful to sterilize titanium devices within 72 h. This in vitro study showed that combined treatment with clarithromycin plus daptomycin is useful to eradicate staphylococcal biofilms formed on orthopedic devices. PMID:26162777

  2. Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

    PubMed

    Cattelan, Natalia; Villalba, María Inés; Parisi, Gustavo; Arnal, Laura; Serra, Diego Omar; Aguilar, Mario; Yantorno, Osvaldo

    2016-02-01

    Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition. PMID:26673448

  3. Evaluation of Multidrug Resistant Staphylococcus aureus and their Association with Biofilm Production in a Tertiary Care Hospital, Tripura, Northeast India

    PubMed Central

    Bir, Raunak; Majumdar, Tapan

    2015-01-01

    Background High morbidity and mortality rates are associated with Methicillin-resistant Staphylococcus aureus (MRSA) because of development of multidrug resistance. Staphylococcus aureus (S. aureus) has the ability to colonize and form biofilms on biomaterials which is causing resistance towards antimicrobials and thus making them difficult to eradicate from the infected hosts. Materials and Methods Culture isolation, identification was done following standard protocol and antibiogram of the isolates were done. The detection of MRSA, Macrolide-Lincosamide-Streptogramin B resistance (MLSB), vancomycin resistance phenotypes were done by using cefoxitin disc diffusion test, D zone test and vancomycin E test. Biofilm was detected by Congo red agar method. Results A total of 100 (31.7%) S. aureus strains were isolated from 315 clinical specimens. The prevalence of MRSA was 47% (47/100) with 85.1% were homogeneous MRSA and 14.9% were heterogeneous. Out of 47 MRSA strains, 63.8% were Hospital acquired-MRSA (HA-MRSA) infections whereas rests 36.2% were caused by Community acquired-MRSA (CA-MRSA) strains. Maximum number of MRSA isolates belonged to group A biotype (34%). A 14.9% isolates were of nontypeable group. Out of 100 S. aureus isolates, the prevalence of Vancomycin resistant S. aureus (VRSA) was found to be 3%. The MLSB phenotypes showed that the rates of inducible MLSB (iMLSB), constitutive MLSB (cMLSB) and Macrolide-Streptogramin B (MSB) in case of MRSA to be 19.1%, 31.9% and 12.8%. Prevalence of low-level (MUPL) and high-level mupirocin resistance (MUPH) among MRSA was 19.1% and 6.4%. Biofilm production was found in 55% strains of S. aureus. Out of 47 MRSA strains 76.6%were producing biofilm in comparison to 38.8% in methicillin-sensitive S. aureus (MSSA). Higher degree of antibiotic resistance in biofilm producers was seen especially in case of ciprofloxacin, co-trimoxazole, rifampicin, kanamycin, erythromycin and clindamycin whereas gentamycin, tetracycline and

  4. The inactivation of Staphylococcus aureus biofilms using low-power argon plasma in a layer-by-layer approach

    PubMed Central

    Traba, Christian; Liang, Jun F.

    2014-01-01

    The direct application of low power argon plasma for the decontamination of pre-formed Staphylococcus aureus biofilms on various surfaces was examined. Distinct chemical/physical properties of reactive species found in argon plasmas generated at different wattages all demonstrated very potent but very different anti-biofilm mechanisms of action. An in depth analysis of results showed that: (1) the different reactive species produced in each plasma demonstrated specific antibacterial and/or anti-biofilm activity, and 2) the commonly associated etching effect could be manipulated and even controlled, depending on experimental conditions. Under optimal experimental parameters, bacterial cells in S. aureus biofilms were killed (>99.9%) by plasmas within 10 min of exposure and no bacteria nor biofilm re-growth from argon discharge gas treated biofilms was observed for 150 h. The decontamination ability of plasmas for the treatment of biofilm related contaminations on various materials was confirmed and an entirely novel layer-by-layer decontamination approach was designed and examined. PMID:25569189

  5. The inactivation of Staphylococcus aureus biofilms using low-power argon plasma in a layer-by-layer approach.

    PubMed

    Traba, Christian; Liang, Jun F

    2015-01-01

    The direct application of low power argon plasma for the decontamination of pre-formed Staphylococcus aureus biofilms on various surfaces was examined. Distinct chemical/physical properties of reactive species found in argon plasmas generated at different wattages all demonstrated very potent but very different anti-biofilm mechanisms of action. An in-depth analysis of the results showed that: (1) the different reactive species produced in each plasma demonstrated specific antibacterial and/or anti-biofilm activity; and (2) the commonly associated etching effect could be manipulated and even controlled, depending on the experimental conditions. Under optimal experimental parameters, bacterial cells in S. aureus biofilms were killed (> 99.9%) by plasmas within 10 min of exposure and no bacteria nor biofilm regrowth from argon discharge gas treated biofilms was observed for 150 h. The decontamination ability of plasmas for the treatment of biofilm related contaminations on various materials was confirmed and an entirely novel layer-by-layer decontamination approach was designed and examined. PMID:25569189

  6. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material.

    PubMed

    Vassena, Christian; Fenu, Simone; Giuliani, Francesco; Fantetti, Lia; Roncucci, Gabrio; Simonutti, Giulio; Romanò, Carlo Luca; De Francesco, Raffaele; Drago, Lorenzo

    2014-07-01

    Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant micro-organisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms. PMID:24933446

  7. inhibitory effects of citral, cinnamaldehyde, and tea polyphenols on mixed biofilm formation by foodborne Staphylococcus aureus and Salmonella enteritidis.

    PubMed

    Zhang, Hongmei; Zhou, Wenyuan; Zhang, Wenyan; Yang, Anlin; Liu, Yanlan; Jiang, Yan; Huang, Shaosong; Su, Jianyu

    2014-06-01

    Biofilms are significant hazards in the food industry. In this study, we investigated the effects of food additive such as citral, cinnamaldehyde, and tea polyphenols on mixed biofilm formation by foodborne Staphylococcus aureus and Salmonella serotype Enteritidis. The adhesion rates of mixed strains in sub-MIC of additives were determined by a microtiter plate assay and bacterial communication signal autoinducer 2 (AI-2) production via a bioluminescence reporter Vibrio harveyi BB170. The structure of mixed biofilm was analyzed using scanning electron microscopy. The effect of the disinfectants hydrogen peroxide, sodium hypochlorite, and peracetic acid was tested on the mixed biofilm. Our results demonstrated that citral, cinnamaldehyde, and tea polyphenols were able to significantly inhibit mixed biofilm formation, while citral could reduce the synthesis of AI-2. Conversely, we observed a significant increase in AI-2 mediated by cinnamaldehyde. Tea polyphenols at lower concentrations induced AI-2 synthesis; however, AI-2 synthesis was significantly inhibited at higher concentrations (300 m g/ml). Food additives inhibited the adhesion of mixed bacteria on stainless steel chips and increased the sensitivity of the mixed biofilm to disinfectants. In conclusion, citral, cinnamaldehyde, and tea polyphenols had strong inhibitory effects on mixed biofilm formation and also enhanced the effect of disinfectant on mixed biofilm formation. This study provides a scientific basis for the application of natural food additives to control biofilm formation of foodborne bacteria. PMID:24853514

  8. Effects of antibiotics on biofilm and unattached cells of a clinical Staphylococcus aureus isolate from bone and joint infection.

    PubMed

    Marquès, Claire; Tasse, Jason; Pracros, Anne; Collin, Valérie; Franceschi, Christine; Laurent, Frédéric; Chatellier, Sonia; Forestier, Christiane

    2015-09-01

    Treatment of orthopaedic infections remains challenging owing to the inability of antibiotics to eradicate biofilms and prevent their regrowth. The present study characterized the effects of 12 antibiotics on in vitro biofilm formed by a representative strain of meticillin-susceptible Staphylococcus aureus (MSSA) isolated from a bone infection. Determination of the minimum biofilm eradication concentrations indicated that in vitro eradication of 24 h-old biofilms required concentrations up to 51,200 times higher than MICs. The influence of the same panel of antibiotics was also investigated on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the suspensions (individual cells) and the biofilm biomass. Except for fusidic acid, the presence of antibiotics during the initial steps of biofilm formation resulted in significant decreases in the number of sessile viable bacteria at the highest concentrations tested. Ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin were the most effective drugs. Confocal microscopy analysis indicated that daptomycin was more efficient at bacteria lysis than gentamicin and vancomycin. However, viable individual cells were still detectable in the assays performed with ceftarolin, fosfomycin, ofloxacin, rifampicin and vancomycin at concentrations for which no sessile cells were detected. Although none of the molecules tested was effective at classical therapeutic concentrations against 24 h-old MSSA biofilms, all except fusidic acid were able to impair biofilm formation at concentrations near the breakpoints. However, presence of viable individual unattached cells could imply a significant risk of microbial dissemination and increased risk of infections. PMID:26297246

  9. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of swine origin form robust biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methicillin-resistant Staphylococcus aureus (MRSA) colonization of livestock animals is common and prevalence rates for pigs have been reported to be as high as 49%. One hypothesis to explain the high prevalence of MRSA in swine herds is the ability of these organisms to exist as biofilms. To invest...

  10. Biofilm formation by Staphylococcus aureus from food contact surfaces in a meat-based broth and sensitivity to sanitizers.

    PubMed

    de Souza, Evandro Leite; Meira, Quênia Gramile Silva; de Medeiros Barbosa, Isabella; Athayde, Ana Júlia Alves Aguiar; da Conceição, Maria Lúcia; de Siqueira Júnior, José Pinto

    2014-01-01

    This study assessed the capacity of adhesion, the detachment kinetic and the biofilm formation by Staphylococcus aureus isolated from food services on stainless steel and polypropylene surfaces (2 × 2 cm) when cultivated in a meat-based broth at 28 and 7 °C. It was also to study the efficacy of the sanitizers sodium hypochlorite (250 mg/L) and peracetic acid (30 mg/L) in inactivating the bacterial cells in the preformed biofilm. S. aureus strains adhered in high numbers regardless the assayed surface kind and incubation temperature over 72 h. Cells detachment of surfaces revealed high persistence over the incubation period. Number of cells needed for biofilm formation was noted at all experimental systems already after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered on polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacity to adhere and form biofilm on polypropylene and stainless steel surfaces under different growth conditions. Moreover, the cells in biofilm matrix were resistant for total removal when submitted to the exposure to sanitizers. PMID:24948915

  11. Biofilm formation by Staphylococcus aureus from food contact surfaces in a meat-based broth and sensitivity to sanitizers

    PubMed Central

    de Souza, Evandro Leite; Meira, Quênia Gramile Silva; de Medeiros Barbosa, Isabella; Athayde, Ana Júlia Alves Aguiar; da Conceição, Maria Lúcia; de Siqueira Júnior, José Pinto

    2014-01-01

    This study assessed the capacity of adhesion, the detachment kinetic and the biofilm formation by Staphylococcus aureus isolated from food services on stainless steel and polypropylene surfaces (2 × 2 cm) when cultivated in a meat-based broth at 28 and 7 °C. It was also to study the efficacy of the sanitizers sodium hypochlorite (250 mg/L) and peracetic acid (30 mg/L) in inactivating the bacterial cells in the preformed biofilm. S. aureus strains adhered in high numbers regardless the assayed surface kind and incubation temperature over 72 h. Cells detachment of surfaces revealed high persistence over the incubation period. Number of cells needed for biofilm formation was noted at all experimental systems already after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered on polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacity to adhere and form biofilm on polypropylene and stainless steel surfaces under different growth conditions. Moreover, the cells in biofilm matrix were resistant for total removal when submitted to the exposure to sanitizers. PMID:24948915

  12. Discovery of quinoline small molecules with potent dispersal activity against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a scaffold hopping strategy.

    PubMed

    Abouelhassan, Yasmeen; Garrison, Aaron T; Burch, Gena M; Wong, Wilson; Norwood, Verrill M; Huigens, Robert W

    2014-11-01

    Staphylococcus aureus and Staphylococcus epidermidis are recognized as the most frequent cause of biofilm-associated nosocomial and indwelling medical device infections. Biofilm-associated infections are known to be highly resistant to our current arsenal of clinically used antibiotics and antibacterial agents. To exacerbate this problem, no therapeutic option exists that targets biofilm-dependent machinery critical to Staphylococcal biofilm formation and maintenance. Here, we describe the discovery of a series of quinoline small molecules that demonstrate potent biofilm dispersal activity against methicillin-resistant S. aureus and S. epidermidis using a scaffold hopping strategy. This interesting class of quinolines also has select synthetic analogues that demonstrate potent antibacterial activity and biofilm inhibition against S. aureus and S. epidermidis. PMID:25264073

  13. Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections.

    PubMed

    Rohde, Holger; Burandt, Eike C; Siemssen, Nicolaus; Frommelt, Lars; Burdelski, Christoph; Wurster, Sabine; Scherpe, Stefanie; Davies, Angharad P; Harris, Llinos G; Horstkotte, Matthias A; Knobloch, Johannes K-M; Ragunath, Chandran; Kaplan, Jeffrey B; Mack, Dietrich

    2007-03-01

    Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules. PMID:17187854

  14. An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections.

    PubMed

    Zapotoczna, Marta; McCarthy, Hannah; Rudkin, Justine K; O'Gara, James P; O'Neill, Eoghan

    2015-12-15

    High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection. PMID:26044292

  15. Low Fluid Shear Culture of Staphylococcus Aureus Represses hfq Expression and Induces an Attachment-Independent Biofilm Phenotype

    NASA Technical Reports Server (NTRS)

    Ott, C. Mark; Castro, S. L.; Nickerson, C. A.; Nelman-Gonzalez, M.

    2011-01-01

    Background: The opportunistic pathogen, Staphylococcus aureus, experiences fluctuations in fluid shear during infection and colonization of a human host. Colonization frequently occurs at mucus membrane sites such as in the gastrointestinal tract where the bacterium may experience low levels of fluid shear. The response of S. aureus to low fluid shear remains unclear. Methods: S. aureus was cultured to stationary phase using Rotating-Wall Vessel (RWV) bioreactors which produce a physiologically relevant low fluid shear environment. The bacterial aggregates that developed in the RWV were evaluated by electron microscopy as well as for antibiotic resistance and other virulence-associated stressors. Genetic expression profiles for the low-shear cultured S. aureus were determined by microarray analysis and quantitative real-time PCR. Results: Planktonic S. aureus cultures in the low-shear environment formed aggregates completely encased in high amounts of extracellular polymeric substances. In addition, these aggregates demonstrated increased antibiotic resistance indicating attachment-independent biofilm formation. Carotenoid production in the low-shear cultured S. aureus was significantly decreased, and these cultures displayed an increased susceptibility to oxidative stress and killing by whole blood. The hfq gene, associated with low-shear growth in Gram negative organisms, was also found to be down-regulated in S. aureus. Conclusions: Collectively, this data suggests that S. aureus decreases virulence characteristics in favor of a biofilm-dwelling colonization phenotype in response to a low fluid shear environment. Furthermore, the identification of an Hfq response to low-shear culture in S. aureus, in addition to the previously reported responses in Gram negative organisms, strongly suggests an evolutionarily conserved response to mechanical stimuli among structurally diverse prokaryotes.

  16. The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development.

    PubMed

    Malamud, Florencia; Torres, Pablo S; Roeschlin, Roxana; Rigano, Luciano A; Enrique, Ramón; Bonomi, Hernán R; Castagnaro, Atilio P; Marano, María Rosa; Vojnov, Adrián A

    2011-03-01

    Xanthomonas axonopodis pv. citri (Xac) is the causative agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. To evaluate the participation of the single flagellum of Xac in biofilm formation, mutants in the fliC (flagellin) and the flgE (hook) genes were generated. Swimming motility, assessed on 0.25 % agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellar-independent surface translocation on 0.5 % agar plates. Mutation of either the rpfF or the rpfC gene, which both encode proteins involved in cell-cell signalling mediated by diffusible signal factor (DSF), led to a reduction in both flagellar-dependent and flagellar-independent surface translocation, indicating a regulatory role for DSF in both types of motility. Confocal laser scanning microscopy of biofilms produced in static culture demonstrated that the flagellum is also involved in the formation of mushroom-shaped structures and water channels, and in the dispersion of biofilms. The presence of the flagellum was required for mature biofilm development on lemon leaf surfaces. The absence of flagellin produced a slight reduction in Xac pathogenicity and this reduction was more severe when the complete flagellum structure was absent. PMID:21109564

  17. Impact of biocides on biofilm formation by methicillin-resistant Staphylococcus aureus (ST239-SCCmecIII) isolates.

    PubMed

    Aparecida Guimarães, Marcia; Rocchetto Coelho, Leonardo; Rodrigues Souza, Raquel; Ferreira-Carvalho, Bernadete Teixeira; Marie Sá Figueiredo, Agnes

    2012-03-01

    Procedures of sterilization and disinfection are essential to ensure that medical and surgical instruments will not transmit infectious pathogens to patients. In the present paper, we tested the residual effect of these compounds on biofilm formation and its efficiency in disrupting preformed biofilms using methicillin-resistant Staphylococcus aureus (MRSA) isolates of the lineage ST239-SCCmecIII. All compounds examined, except 70% alcohol, caused a significant impairment in biofilm formation with concomitant inhibition of cell growth. Among the compounds examined, 10% povidone-iodine (PVP-I) was the only antiseptic that exhibited more than 90% reduction of both biofilm formation and dispersion. In the group of sterilants and disinfectants, a formulation containing 7% hydrogen peroxide and 0.2% peracetic acid (HP-PA), and sodium hypochlorite with 1% active chlorine (NaOCl) were equally effective. PMID:22211887

  18. Nfu facilitates the maturation of iron-sulfur proteins and participates in virulence in Staphylococcus aureus

    PubMed Central

    Mashruwala, Ameya A.; Pang, Yun Y.; Rosario-Cruz, Zuelay; Chahal, Harsimranjit K.; Benson, Meredith A.; Anzaldi-Mike, Laura L.; Skaar, Eric P.; Torres, Victor J.; Nauseef, William M.; Boyd, Jeffrey M.

    2015-01-01

    Summary The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron-sulfur (Fe-S) clusters, which are required for functional Fe-S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe-S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe-S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as a Fe-S cluster carrier, which aids in the maturation of Fe-S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non-incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe-S cluster metabolism as an attractive antimicrobial target. PMID:25388433

  19. Investigation of the antibiotic resistance and biofilm formation of Staphylococcus aureus strains isolated from gangrenous mastitis of ewes.

    PubMed

    Tel, Osman Yaşar; Aslantaş, Ozkan; Keskin, Oktay; Yilmaz, Ebru Sebnem; Demir, Cemil

    2012-06-01

    In this study, Staphylococcus aureus strains (n = 110) isolated from seven ewe flocks in Sanliurfa, Turkey were screened for antibiotic resistance and biofilmforming ability as well as for genes associated with antibiotic resistance and biofilm-forming ability. All isolates were found to be susceptible to oxacillin, gentamicin, clindamycin, cefoxitin, tetracycline, vancomycin, amoxicillin-clavulanic acid, ciprofloxacin and sulphamethoxazole-trimethoprim. The percent proportions of strains resistant to penicillin G, ampicillin and erythromycin were 27.2% (n = 30), 25.4% (n = 28) and 6.3% (n = 7), respectively. Regarding the antibiotic resistance genes, 32 (29%) isolates carried the blaZ and 8 (7.2%) the ermC gene. Other resistance genes were not detected in the isolates. All isolates showed biofilm-forming ability on Congo red agar (CRA), while 108 (98.18%) and 101 (91.81%) of them were identified as biofilm producers by the use of standard tube (ST) and microplate (MP) methods, respectively. All isolates carried the icaA and icaD genes but none of them harboured the bap gene. The results demonstrated that S. aureus isolates from gangrenous mastitis were mainly resistant to penicillins (which are susceptible to the staphylococcal beta-lactamase enzyme), and less frequently to erythromycin. Furthermore, all of the S. aureus isolates produced biofilm which was considered a potential virulence factor in the pathogenesis of staphylococcal mastitis. PMID:22609990

  20. Advantage of Upregulation of Succinate Dehydrogenase in Staphylococcus aureus Biofilms

    PubMed Central

    Gaupp, Rosmarie; Schlag, Steffen; Liebeke, Manuel; Lalk, Michael; Götz, Friedrich

    2010-01-01

    Previous studies have demonstrated that various tricarboxylic acid (TCA) cycle genes, particularly the succinate dehydrogenase genes (sdhCAB), are upregulated in Staphylococcus aureus biofilms. To better study the role of this enzyme complex, an sdhCAB deletion mutant (Δsdh) was constructed. Compared to the wild type (wt) the mutant was impaired in planktonic growth under aerobic conditions, excreted acetic acid could not be reused and accumulated continuously, succinate was excreted and found in the culture supernatant, and metabolome analysis with cells grown in chemically defined medium revealed reduced uptake/metabolism of some amino acids from the growth medium. Moreover, the mutant was able to counteract the steadily decreasing extracellular pH by increased urease activity. The addition of fumarate to the growth medium restored the wt phenotype. The mutant showed a small-colony variant (SCV)-like phenotype, a slight increase in resistance to various aminoglycoside antibiotics, and decreased pigmentation. The decreased growth under aerobic conditions is due to the interruption of the TCA cycle (indicated by the accumulation of succinate and acetic acid) with the consequence that many fewer reduction equivalents (NADH and FADH2) can fuel the respiratory chain. The results indicate that the TCA cycle is required for acetate and amino acid catabolism; its upregulation under biofilm conditions is advantageous under such nutrient- and oxygen-limited conditions. PMID:20207757

  1. Vaccine development in Staphylococcus aureus: taking the biofilm phenotype into consideration

    PubMed Central

    Harro, Janette M; Peters, Brian M; O'May, Graeme A; Archer, Nathan; Kerns, Patrick; Prabhakara, Ranjani; Shirtliff, Mark E

    2010-01-01

    Vaccine development against pathogenic bacteria is an imperative initiative as bacteria are gaining resistance to current antimicrobial therapies and few novel antibiotics are being developed. Candidate antigens for vaccine development can be identified by a multitude of high-throughput technologies that were accelerated by access to complete genomes. While considerable success has been achieved in vaccine development against bacterial pathogens, many species with multiple virulence factors and modes of infection have provided reasonable challenges in identifying protective antigens. In particular, vaccine candidates should be evaluated in the context of the complex disease properties, whether planktonic (e.g. sepsis and pneumonia) and/or biofilm associated (e.g. indwelling medical device infections). Because of the phenotypic differences between these modes of growth, those vaccine candidates chosen only for their efficacy in one disease state may fail against other infections. This review will summarize the history and types of bacterial vaccines and adjuvants as well as present an overview of modern antigen discovery and complications brought about by polymicrobial infections. Finally, we will also use one of the better studied microbial species that uses differential, multifactorial protein profiles to mediate an array of diseases, Staphylococcus aureus, to outline some of the more recently identified problematic issues in vaccine development in this biofilm-forming species. PMID:20602638

  2. Dynamics of Biofilm Formation and the Interaction between Candida albicans and Methicillin-Susceptible (MSSA) and -Resistant Staphylococcus aureus (MRSA)

    PubMed Central

    Zago, Chaiene Evelin; Silva, Sónia; Sanitá, Paula Volpato; Barbugli, Paula Aboud; Dias, Carla Maria Improta; Lordello, Virgínia Barreto; Vergani, Carlos Eduardo

    2015-01-01

    Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms. PMID:25875834

  3. Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

    PubMed Central

    Oyama, Takuto; Miyazaki, Motoyasu; Yoshimura, Michinobu; Takata, Tohru; Ohjimi, Hiroyuki; Jimi, Shiro

    2016-01-01

    Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA). MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF) and low-biofilm formers (L-BF). These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections. PMID:27376326

  4. Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice.

    PubMed

    Oyama, Takuto; Miyazaki, Motoyasu; Yoshimura, Michinobu; Takata, Tohru; Ohjimi, Hiroyuki; Jimi, Shiro

    2016-01-01

    Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA). MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF) and low-biofilm formers (L-BF). These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections. PMID:27376326

  5. Efficacy of Linezolid and Fosfomycin in Catheter-Related Biofilm Infection Caused by Methicillin-Resistant Staphylococcus aureus.

    PubMed

    Chai, Dong; Liu, Xu; Wang, Rui; Bai, Yan; Cai, Yun

    2016-01-01

    As long-standing clinical problems, catheter-related infections and other chronic biofilm infections are more difficult to treat due to the high antibiotic resistance of biofilm. Therefore, new treatments are needed for more effective bacteria clearance. In this study, we evaluated the antibacterial activities of several common antibiotics alone and their combinations against biofilm-embedded methicillin-resistant staphylococcus aureus (MRSA) infections, both in vitro and in vivo. In brief, fosfomycin, levofloxacin, and rifampin alone or in combination with linezolid were tested in vitro against planktonic and biofilm-embedded MRSA infection in three MRSA stains. The synergistic effects between linezolid and the other three antibiotics were assessed by fractional inhibitory concentration index (FICI) and time-kill curves, where the combination of linezolid plus fosfomycin showed the best synergistic effect in all strains. For further evaluation in vivo, we applied the combination of linezolid and fosfomycin in a catheter-related biofilm rat model and found that viable bacteria counts in biofilm were significantly reduced after treatment (P < 0.05). In summary, we have shown here that the combination of linezolid and fosfomycin treatment had improved therapeutic effects on biofilm-embedded MRSA infection both in vitro and in vivo, which provided important basis for new clinical therapy development. PMID:27366751

  6. Efficacy of Linezolid and Fosfomycin in Catheter-Related Biofilm Infection Caused by Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Chai, Dong; Liu, Xu; Wang, Rui; Bai, Yan; Cai, Yun

    2016-01-01

    As long-standing clinical problems, catheter-related infections and other chronic biofilm infections are more difficult to treat due to the high antibiotic resistance of biofilm. Therefore, new treatments are needed for more effective bacteria clearance. In this study, we evaluated the antibacterial activities of several common antibiotics alone and their combinations against biofilm-embedded methicillin-resistant staphylococcus aureus (MRSA) infections, both in vitro and in vivo. In brief, fosfomycin, levofloxacin, and rifampin alone or in combination with linezolid were tested in vitro against planktonic and biofilm-embedded MRSA infection in three MRSA stains. The synergistic effects between linezolid and the other three antibiotics were assessed by fractional inhibitory concentration index (FICI) and time-kill curves, where the combination of linezolid plus fosfomycin showed the best synergistic effect in all strains. For further evaluation in vivo, we applied the combination of linezolid and fosfomycin in a catheter-related biofilm rat model and found that viable bacteria counts in biofilm were significantly reduced after treatment (P < 0.05). In summary, we have shown here that the combination of linezolid and fosfomycin treatment had improved therapeutic effects on biofilm-embedded MRSA infection both in vitro and in vivo, which provided important basis for new clinical therapy development. PMID:27366751

  7. In vitro pharmacokinetics of antimicrobial cationic peptides alone and in combination with antibiotics against methicillin resistant Staphylococcus aureus biofilms.

    PubMed

    Dosler, Sibel; Mataraci, Emel

    2013-11-01

    Antibiotic therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming more difficult in hospitals and communities because of strong biofilm-forming properties and multidrug resistance. Biofilm-associated MRSA is not affected by therapeutically achievable concentrations of antibiotics. Therefore, we investigated the in vitro pharmacokinetic activities of antimicrobial cationic peptides (AMPs; indolicidin, cecropin [1-7]-melittin A [2-9] amide [CAMA], and nisin), either alone or in combination with antibiotics (daptomycin, linezolid, teicoplanin, ciprofloxacin, and azithromycin), against standard and 2 clinically obtained MRSA biofilms. The minimum inhibitory concentrations (MIC) and minimum biofilm-eradication concentrations (MBEC) were determined by microbroth dilution technique. The time-kill curve (TKC) method was used to determine the bactericidal activities of the AMPs alone and in combination with the antibiotics against standard and clinically obtained MRSA biofilms. The MIC values of the AMPs and antibiotics ranged between 2 to 16 and 0.25 to 512 mg/L, and their MBEC values were 640 and 512 to 5120 mg/L, respectively. The TKC studies demonstrated that synergistic interactions occurred most frequently when using nisin+daptomycin/ciprofloxacin, indolicidin+teicoplanin, and CAMA+ciprofloxacin combinations. No antagonism was observed with any combination. AMPs appear to be good candidates for the treatment of MRSA biofilms, as they act as both enhancers of anti-biofilm activities and help to prevent or delay the emergence of resistance when used either alone or in combination with antibiotics. PMID:23988790

  8. Effects of extracts from Italian medicinal plants on planktonic growth, biofilm formation and adherence of methicillin-resistant Staphylococcus aureus

    PubMed Central

    Quave, Cassandra L.; Plano, Lisa R.W.; Pantuso, Traci; Bennett, Bradley C.

    2008-01-01

    One-third of botanical remedies from southern Italy are used to treat skin and soft tissue infection (SSTI). Staphylococcus aureus, a common cause of SSTI, has generated increasing concern due to drug resistance. Many plants possess antimicrobial agents and provide effective remedies for SSTI. Our aim was to investigate plants from different ethnobotanical usage groups for inhibition of growth and biofilms in methicillin-resistant S. aureus (MRSA). Three groups were assessed: plant remedies for SSTI, plant remedies not involving the skin, and plants with no ethnomedical application. We screened 168 extracts, representing 104 botanical species, for activity against MRSA (ATCC 33593). We employed broth dilution methods to determine the MIC after 18 hours growth using an optical density (OD600nm) reading. Anti-biofilm effects were assessed by growing biofilms for 40 hours, then fixing and staining with crystal violet. After washing, 10% Tween 80 was added and OD570nm readings were taken. Extracts from 10 plants exhibited an IC50 ≤32 μg/ml for biofilm inhibition: Lonicera alpigena, Castanea sativa, Juglans regia, Ballota nigra, Rosmarinus officinalis, Leopoldia comosa, Malva sylvestris, Cyclamen hederifolium, Rosa canina, and Rubus ulmifolius. Limited bacteriostatic activity was evident. The anti-biofilm activity of medicinal plants was significantly greater than plants without any ethnomedical applications. PMID:18556162

  9. Synergistic antibiofilm efficacy of various commercial antiseptics, enzymes and EDTA: a study of Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

    PubMed

    Lefebvre, Elodie; Vighetto, Christophe; Di Martino, Patrick; Larreta Garde, Véronique; Seyer, Damien

    2016-08-01

    A multistep strategy was used to generate a combined antibiofilm treatment that could efficiently decrease the biomass of dense biofilms (≥6 × 10(7) CFU/cm(2)). Several compounds that exhibited activity against various targets were tested individually and in combination to search for possible synergistic effects. First, the antibiofilm activity of various commercially available antiseptics was tested on Pseudomonas aeruginosa and Staphylococcus aureus. Second, antiseptics were mixed with ethylene diamine tetra-acetic acid (EDTA), which is an ion chelator that can disturb biofilm organisation, and additive effects on biofilm biomass degradation were found for both strains. Then, enzymes with the ability to destabilise the biofilm matrix by hydrolysing either its proteins or its polysaccharides were used; as expected, they did not decrease bacterial viability but were revealed as efficient biomass reducers. The combination of antiseptics, EDTA and proteases, all at low concentrations, revealed a synergistic effect leading to total eradication of dense biofilms both of P. aeruginosa and S. aureus. PMID:27424598

  10. d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

    PubMed Central

    Akers, Kevin S.; Romano, Desiree R.; Woodbury, Ronald L.; Hardy, Sharanda K.; Murray, Clinton K.; Wenke, Joseph C.

    2014-01-01

    Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of d-amino acids (d-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of d-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. d-Met, d-Phe, and d-Trp at concentrations of ≥5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (d-Met/d-Phe/d-Trp). When combined with d-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of d-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of d-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity. PMID:24841260

  11. Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model

    PubMed Central

    Lemmens-den Toom, N. A.; Willemse, J.; Koning, R. A.; Demmers, J. A. A.; Dekkers, D. H. W.; Rijkers, E.; El Ghalbzouri, A.; Nibbering, P. H.; van Wamel, W.

    2016-01-01

    Background & Aim The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. Results All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Conclusion Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections. PMID:26741798

  12. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics.

    PubMed

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the 'holy grail' in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  13. Efficiency of vanilla, patchouli and ylang ylang essential oils stabilized by iron oxide@C14 nanostructures against bacterial adherence and biofilms formed by Staphylococcus aureus and Klebsiella pneumoniae clinical strains.

    PubMed

    Bilcu, Maxim; Grumezescu, Alexandru Mihai; Oprea, Alexandra Elena; Popescu, Roxana Cristina; Mogoșanu, George Dan; Hristu, Radu; Stanciu, George A; Mihailescu, Dan Florin; Lazar, Veronica; Bezirtzoglou, Eugenia; Chifiriuc, Mariana Carmen

    2014-01-01

    Biofilms formed by bacterial cells are associated with drastically enhanced resistance against most antimicrobial agents, contributing to the persistence and chronicization of the microbial infections and to therapy failure. The purpose of this study was to combine the unique properties of magnetic nanoparticles with the antimicrobial activity of three essential oils to obtain novel nanobiosystems that could be used as coatings for catheter pieces with an improved resistance to Staphylococcus aureus and Klebsiella pneumoniae clinical strains adherence and biofilm development. The essential oils of ylang ylang, patchouli and vanilla were stabilized by the interaction with iron oxide@C14 nanoparticles to be further used as coating agents for medical surfaces. Iron oxide@C14 was prepared by co-precipitation of Fe+2 and Fe+3 and myristic acid (C14) in basic medium. Vanilla essential oil loaded nanoparticles pelliculised on the catheter samples surface strongly inhibited both the initial adherence of S. aureus cells (quantified at 24 h) and the development of the mature biofilm quantified at 48 h. Patchouli and ylang-ylang essential oils inhibited mostly the initial adherence phase of S. aureus biofilm development. In the case of K. pneumoniae, all tested nanosystems exhibited similar efficiency, being active mostly against the adherence K. pneumoniae cells to the tested catheter specimens. The new nanobiosystems based on vanilla, patchouli and ylang-ylang essential oils could be of a great interest for the biomedical field, opening new directions for the design of film-coated surfaces with anti-adherence and anti-biofilm properties. PMID:25375335

  14. Darwinolide, a New Diterpene Scaffold That Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm from the Antarctic Sponge Dendrilla membranosa.

    PubMed

    von Salm, Jacqueline L; Witowski, Christopher G; Fleeman, Renee M; McClintock, James B; Amsler, Charles D; Shaw, Lindsey N; Baker, Bill J

    2016-06-01

    A new rearranged spongian diterpene, darwinolide, has been isolated from the Antarctic Dendroceratid sponge Dendrilla membranosa. Characterized on the basis of spectroscopic and crystallographic analysis, the central seven-membered ring is hypothesized to originate from a ring-expansion of a spongian precursor. Darwinolide displays 4-fold selectivity against the biofilm phase of methicillin-resistant Staphylococcus aureus compared to the planktonic phase and may provide a scaffold for the development of therapeutics for this difficult to treat infection. PMID:27175857

  15. Darwinolide, a New Diterpene Scaffold That Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm from the Antarctic Sponge Dendrilla membranosa

    PubMed Central

    2016-01-01

    A new rearranged spongian diterpene, darwinolide, has been isolated from the Antarctic Dendroceratid sponge Dendrilla membranosa. Characterized on the basis of spectroscopic and crystallographic analysis, the central seven-membered ring is hypothesized to originate from a ring-expansion of a spongian precursor. Darwinolide displays 4-fold selectivity against the biofilm phase of methicillin-resistant Staphylococcus aureus compared to the planktonic phase and may provide a scaffold for the development of therapeutics for this difficult to treat infection. PMID:27175857

  16. Development of an intracanal mature Enterococcus faecalis biofilm and its susceptibility to some antimicrobial intracanal medications; an in vitro study

    PubMed Central

    Saber, Shehab El-Din Mohamed; El-Hady, Soha A.

    2012-01-01

    Objectives: To develop a mature biofilm of Enterococcus faecalis inside the root canal system and to test its susceptibility to some antimicrobial medications in vitro. Methods: Single rooted premolars were mechanically enlarged, sterilized, and then infected with a clinical isolate of E. faecalis. Biofilm formation and maturation was monitored using SEM. Biofilm bacteria were exposed to Amoxicillin+clavulanate, Ciprofloxacin, Clindamycin, Doxycycline, and calcium hydroxide as intracanal medications for 1 week. Finally bacterial samples were collected, and colony-forming units were enumerated. Results: SEM examination confirmed the formation of a mature biofilm at the end of the incubation period. All the chemotherapeutic agents used were significantly better than Calcium hydroxide in elimination of biofilm bacteria. The antimicrobial effect of Amoxicillin + clavulanate, Ciprofloxacin and Clindamycin was significantly better than Doxycycline (P=.05). However the difference in the antimicrobial effectiveness among them was statistically non-significant (P=.05). Conclusions: The method used for bacterial biofilm development and maturation is reliable and can be used to assess the anti bacterial potential of endodontic materials. Also, the local application of antibacterial agents can be beneficial in resistant cases of apical periodontitis but only after careful culture and sensitivity testing to choose the appropriate agent for the existing flora. PMID:22229006

  17. Synergistic Photothermal and Antibiotic Killing of Biofilm-Associated Staphylococcus aureus Using Targeted Antibiotic-Loaded Gold Nanoconstructs

    PubMed Central

    2016-01-01

    Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as “ESKAPE pathogens” on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections. PMID

  18. Detection of biofilm related genes, classical enterotoxin genes and agr typing among Staphylococcus aureus isolated from bovine with subclinical mastitis in southwest of Iran.

    PubMed

    Khoramrooz, Seyed Sajjad; Mansouri, Fariba; Marashifard, Masoud; Malek Hosseini, Seyed Ali Asghar; Akbarian Chenarestane-Olia, Fereshteh; Ganavehei, Banafsheh; Gharibpour, Farzaneh; Shahbazi, Ardavan; Mirzaii, Mehdi; Darban-Sarokhalil, Davood

    2016-08-01

    Staphylococcus aureus by producing biofilm and facilitating chronic infection is a common cause of mastitis in cows and thereby can cause food poisoning by production of enterotoxins in milk. The agr typing method is an important tool for epidemiological investigation about S. aureus. The aims of the present study were to detect biofilm related genes, 5 classical enterotoxin genes and the agr types among S. aureus isolates. The ability of S. aureus isolates to produce biofilm was evaluated by modified CRA plate. Six biofilm related adhesion genes (icaD, icaA, fnbA, bap, clfA and cna), five classical enterotoxin genes (sea, seb, sec, sed and see) and tst-1 gene were detected by PCR methods. Multiplex-PCR was used to determination of the agr groups. 55 out of 80(68.8%) S. aureus isolates were biofilm producer. The icaD gene was detected in 70 (87.5%) of isolates. The prevalence rates of fnbA, icaA, clfA, cna and bap were 72.5, 56.25, 50, 22.5, and 5% respectively. The agr group I and III were detected in 57.5% 25% of studied isolates. The sea, sed and tst-1 genes were found in 10%, 7.5% and 1.25% of isolates respectively. The majority of S. aureus were able to produce biofilm. Significant associations were observed between presence of the icaD, icaA, fnbA, clfA and the cna genes as well as biofilm formation. The present study revealed that isolates with the agr type III are more potent for biofilm production. Our data supported a possible link between the agr types and certain SE genes. PMID:27251096

  19. The Quorum Sensing Inhibitor Hamamelitannin Increases Antibiotic Susceptibility of Staphylococcus aureus Biofilms by Affecting Peptidoglycan Biosynthesis and eDNA Release

    PubMed Central

    Brackman, Gilles; Breyne, Koen; De Rycke, Riet; Vermote, Arno; Van Nieuwerburgh, Filip; Meyer, Evelyne; Van Calenbergh, Serge; Coenye, Tom

    2016-01-01

    Treatment of Staphylococcus aureus infections has become increasingly challenging due to the rapid emergence and dissemination of methicillin-resistant strains. In addition, S. aureus reside within biofilms at the site of infection. Few novel antibacterial agents have been developed in recent years and their bacteriostatic or bactericidal activity results in selective pressure, inevitably inducing antimicrobial resistance. Consequently, innovative antimicrobials with other modes of action are urgently needed. One alternative approach is targeting the bacterial quorum sensing (QS) system. Hamamelitannin (2′,5-di-O-galloyl-d-hamamelose; HAM) was previously suggested to block QS through the TraP QS system and was shown to increase S. aureus biofilm susceptibility towards vancomycin (VAN) although mechanistic insights are still lacking. In the present study we provide evidence that HAM specifically affects S. aureus biofilm susceptibility through the TraP receptor by affecting cell wall synthesis and extracellular DNA release of S. aureus. We further provide evidence that HAM can increase the susceptibility of S. aureus biofilms towards different classes of antibiotics in vitro. Finally, we show that HAM increases the susceptibility of S. aureus to antibiotic treatment in in vivo Caenorhabditis elegans and mouse mammary gland infection models. PMID:26828772

  20. Effects of nisin and lysozyme on growth inhibition and biofilm formation capacity of Staphylococcus aureus strains isolated from raw milk and cheese samples.

    PubMed

    Sudagidan, Mert; Yemenicioğlu, Ahmet

    2012-09-01

    Effects of nisin and lysozyme on growth inhibition and biofilm formation capacity of 25 Staphylococcus aureus strains isolated from raw milk (13 strains) and cheese (12 strains) were studied. Nisin was tested at concentrations between 0.5 and 25 μg/ml; the growth of all strains was inhibited at 25 μg/ml, but the resistances of strains showed a great variation at lower nisin concentrations. In contrast, lysozyme tested at concentrations up to 5.0 mg/ml showed no inhibition on the growth of strains. Nisin used at the growth inhibitory concentration prevented the biofilm formation of strains, but strains continued biofilm formation at subinhibitory nisin concentrations. Lysozyme did not affect the biofilm formation of 19 of the strains, but it caused a considerable activation in the biofilm formation capacity of six strains. Twelve of the strains contained both biofilm-related protease genes (sspA, sspB, and aur) and active proteases; eight of these strains were nisin resistant. These results suggest a potential risk of S. aureus growth and biofilm formation when lysozyme is used in the biopreservation of dairy products. Nisin can be used to control growth and biofilm formation of foodborne S. aureus, unless resistance against this biopreservative develops. PMID:22947470

  1. A Novel Approach Utilizing Biofilm Time-Kill Curves To Assess the Bactericidal Activity of Ceftaroline Combinations against Biofilm-Producing Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Barber, Katie E.; Werth, Brian J.; McRoberts, John P.

    2014-01-01

    Medical device infections frequently require combination therapy. Beta-lactams combined with glycopeptides/lipopeptides are bactericidal against methicillin-resistant Staphylococcus aureus (MRSA). Novel macrowell kill-curve methods tested synergy between ceftaroline or cefazolin plus daptomycin, vancomycin, or rifampin against biofilm-producing MRSA. Ceftaroline combinations demonstrated the most pronounced bacterial reductions. Ceftaroline demonstrated greatest kill with daptomycin (4.02 ± 0.59 log10 CFU/cm2), compared to combination with vancomycin (3.36 ± 0.35 log10 CFU/cm2) or rifampin (2.68 ± 0.61 log10 CFU/cm2). These data suggest that beta-lactam combinations are useful against MRSA biofilms. PMID:24614378

  2. Newly-synthesized chalcones-inhibition of adherence and biofilm formation of methicillin-resistant Staphylococcus aureus

    PubMed Central

    Bozic, Dragana D.; Milenkovic, Marina; Ivkovic, Branka; Cirkovic, Ivana

    2014-01-01

    Biofilm formation and adherence of bacteria to host tissue are one of the most important virulence factors of methicillin-resistant strains of Staphylococcus aureus (MRSA). The number of resistant strains is seriously increasing during the past years and bacteria have become resistant, not only to methicillin, but also to other commonly used antistaphylococcal antibiotics. There is a great need for discovering a novel antimicrobial agent for the treatment of staphylococcal infections. One of the most promising groups of compounds appears to be chalcones. In present study we evaluated the in vitro effect of three newly synthesized chalcones: 1,3- Bis-(2-hydroxy-phenyl)-propenone, 3-(3-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone and 3-(4-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone on glycocalyx production, biofilm formation and adherence to human fibronectin of clinical isolates and laboratory control strain of MRSA (ATCC 43300). Subinhibitory concentrations of the tested compounds reduced the production of glycocalyx, biofilm formation and adherence to human fibronectin of all MRSA strains. Inhibition of biofilm formation was dose dependent and the most effective was 1,3- Bis-(2-hydroxy-phenyl)-propenone. In our study we demonstrated that three newly-synthesized chalcones exhibited significant effect on adherence and biofilm formation of MRSA strains. Chalcones may be considered as promising new antimicrobial agents that can be used for prevention of staphylococcal infections or as adjunct to antibiotics in conventional therapy. PMID:24948943

  3. Disinfection of meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a remote non-thermal gas plasma.

    PubMed

    Cotter, J J; Maguire, P; Soberon, F; Daniels, S; O'Gara, J P; Casey, E

    2011-07-01

    The effective disinfection of hospital surfaces is recognised as an important factor in preventing hospital-acquired infections. The purpose of this study was to quantify the disinfection rate of a novel gas plasma system on clinically relevant biofilms. Clinical isolates of Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus (MRSA) were grown as biofilms on glass surfaces and tested in a disinfection container remote from the plasma source. The strains used in this study were known to produce substantial quantities of biofilm and average log₁₀ counts were 9.0 and 9.1 cfu/cm(2) for S. epidermidis and MRSA respectively. Counts were reduced by between 4 and 4.5 log₁₀ after 1h of exposure for MRSA and S. epidermidis respectively. More prolonged treatment in the case of MRSA biofilms resulted in a 5.5 log₁₀ reduction after 90 min. Biofilm samples were also placed in medical device packaging bags and similar rates of disinfection were observed. PMID:21601949

  4. Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms

    PubMed Central

    Thwaite, Joanne E.; Burt, Rebecca; Laws, Thomas R.; Raguse, Marina; Moeller, Ralf; Webber, Mark A.; Oppenheim, Beryl A.

    2016-01-01

    ABSTRACT The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica. All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm2 to 108 J/cm2). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications. IMPORTANCE Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further

  5. Use of Microfluidic Technology To Analyze Gene Expression during Staphylococcus aureus Biofilm Formation Reveals Distinct Physiological Niches

    PubMed Central

    Moormeier, Derek E.; Endres, Jennifer L.; Mann, Ethan E.; Sadykov, Marat R.; Horswill, Alexander R.; Rice, Kelly C.; Fey, Paul D.

    2013-01-01

    The Staphylococcus aureus cid and lrg operons play significant roles in the control of autolysis and accumulation of extracellular genomic DNA (eDNA) during biofilm development. Although the molecular mechanisms mediating this control are only beginning to be revealed, it is clear that cell death must be limited to a subfraction of the biofilm population. In the present study, we tested the hypothesis that cid and lrg expression varies during biofilm development as a function of changes in the availability of oxygen. To examine cid and lrg promoter activity during biofilm development, fluorescent reporter fusion strains were constructed and grown in a BioFlux microfluidic system, generating time-lapse epifluorescence images of biofilm formation, which allows the spatial and temporal localization of gene expression. Consistent with cid induction under hypoxic conditions, the cid::gfp fusion strain expressed green fluorescent protein predominantly within the interior of the tower structures, similar to the pattern of expression observed with a strain carrying a gfp fusion to the hypoxia-induced promoter controlling the expression of the lactose dehydrogenase gene. The lrg promoter was also expressed within towers but appeared more diffuse throughout the tower structures, indicating that it was oxygen independent. Unexpectedly, the results also demonstrated the existence of tower structures with different expression phenotypes and physical characteristics, suggesting that these towers exhibit different metabolic activities. Overall, the findings presented here support a model in which oxygen is important in the spatial and temporal control of cid expression within a biofilm and that tower structures formed during biofilm development exhibit metabolically distinct niches. PMID:23524683

  6. In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin-resistant Staphylococcus aureus biofilms.

    PubMed

    Mataraci, Emel; Dosler, Sibel

    2012-12-01

    Methicillin-resistant Staphylococcus aureus (MRSA) strains are most often found as hospital- and community-acquired infections. The danger of MRSA infections results from not only the emergence of multidrug resistance but also the occurrence of bacteria that form strong biofilms. We investigated the in vitro activities of antibiotics (daptomycin, linezolid, teichoplanine, azithromycin, and ciprofloxacin) and antimicrobial cationic peptides {AMPs; indolicidin, CAMA [cecropin (1-7)-melittin A (2-9) amide], and nisin} alone or in combination against MRSA ATCC 43300 biofilms. The MICs and minimum biofilm eradication concentrations (MBECs) were determined by the broth microdilution technique. Antibiotic and AMP combinations were assessed using the checkerboard technique. For MRSA planktonic cells, MICs of antibiotics and AMPs ranged between 0.125 and 512 and 8 and 16 mg/liter, respectively, and the MBEC values were between 512 and 5,120 and 640 mg/liter, respectively. With a fractional inhibitory concentration of ≤0.5 as the borderline, synergistic interactions against MRSA biofilms were frequent with almost all antibiotic-antibiotic and antibiotic-AMP combinations. Against planktonic cells, they generally had an additive effect. No antagonism was observed. All of the antibiotics, AMPs, and their combinations were able to inhibit the attachment of bacteria at 1/10 MIC and biofilm formation at 1× MIC. Biofilm-associated MRSA was not affected by therapeutically achievable concentrations of antimicrobial agents. Use of a combination of antimicrobial agents can provide a synergistic effect, which rapidly enhances antibiofilm activity and may help prevent or delay the emergence of resistance. AMPs seem to be good candidates for further investigations in the treatment of MRSA biofilms, alone or in combination with antibiotics. PMID:23070152

  7. Ciprofloxacin-Eluting Nanofibers Inhibits Biofilm Formation by Pseudomonas aeruginosa and a Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Ahire, Jayesh J.; Neveling, Deon P.; Hattingh, Melanie; Dicks, Leon M. T.

    2015-01-01

    Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with hospital-acquired infections and are known to form biofilms. Ciprofloxacin (CIP), which is normally used to treat these infections, is seldom effective in killing cells in a biofilm. This is mostly due to slow or weak penetration of CIP to the core of biofilms. The problem is accentuated by the release of CIP below MIC (minimal inhibitory concentration) levels following a rapid (burst) release. The aim of this study was to develop a drug carrier that would keep CIP above MIC levels for an extended period. Ciprofloxacin was suspended into poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO), and electrospun into nanofibers (CIP-F). All of the CIP was released from the nanofibers within 2 h, which is typical of a burst release. However, 99% of P. aeruginosa PA01 cells and 91% of S. aureus Xen 30 cells (a methicillin-resistant strain) in biofilms were killed when exposed to CIP-F. CIP levels remained above MIC for 5 days, as shown by growth inhibition of the cells in vitro. The nanofibers were smooth in texture with no bead formation, as revealed by scanning electron and atomic force microscopy. A single vibration peak at 1632 cm-1, recorded with Fourier transform infrared spectroscopy, indicated that CIP remained in crystal form when incorporated into PDLLA: PEO. No abnormalities in the histology of MCF-12A breast epithelial cells were observed when exposed to CIP-F. This is the first report of the inhibition of biofilm formation by CIP released from PDLLA: PEO nanofibers. PMID:25853255

  8. Development of a biofilm inhibitor molecule against multidrug resistant Staphylococcus aureus associated with gestational urinary tract infections

    PubMed Central

    Balamurugan, P.; Hema, M.; Kaur, Gurmeet; Sridharan, V.; Prabu, P. C.; Sumana, M. N.; Princy, S. Adline

    2015-01-01

    Urinary Tract Infection (UTI) is a globally widespread human infection caused by an infestation of uropathogens. Eventhough, Escherichia coli is often quoted as being the chief among them, Staphylococcus aureus involvement in UTI especially in gestational UTI is often understated. Staphylococcal accessory regulator A (SarA) is a quorum regulator of S. aureus that controls the expression of various virulence and biofilm phenotypes. Since SarA had been a focussed target for antibiofilm agent development, the study aims to develop a potential drug molecule targeting the SarA of S. aureus to combat biofilm associated infections in which it is involved. In our previous studies, we have reported the antibiofilm activity of SarA based biofilm inhibitor, (SarABI) with a 50% minimum biofilm inhibitory concentration (MBIC50) value of 200 μg/mL against S. aureus associated with vascular graft infections and also the antibiofilm activity of the root ethanolic extracts of Melia dubia against uropathogenic E. coli. In the present study, in silico design of a hybrid molecule composed of a molecule screened from M. dubia root ethanolic extracts and a modified SarA based inhibitor (SarABIM) was undertaken. SarABIM is a modified form of SarABI where the fluorine groups are absent in SarABIM. Chemical synthesis of the hybrid molecule, 4-(Benzylamino)cyclohexyl 2-hydroxycinnamate (henceforth referred to as UTI Quorum-Quencher, UTIQQ) was then performed, followed by in vitro and in vivo validation. The MBIC50 and MBIC90 of UTIQQ were found to be 15 and 65 μg/mL, respectively. Confocal laser scanning microscopy (CLSM) images witnessed biofilm reduction and bacterial killing in either UTIQQ or in combined use of antibiotic gentamicin and UTIQQ. Similar results were observed with in vivo studies of experimental UTI in rat model. So, we propose that the drug UTIQQ would be a promising candidate when used alone or, in combination with an antibiotic for staphylococcal associated UTI. PMID

  9. Listeria monocytogenes Impact on Mature or Old Pseudomonas fluorescens Biofilms During Growth at 4 and 20°C

    PubMed Central

    Puga, Carmen H.; Orgaz, Belen; SanJose, Carmen

    2016-01-01

    Changes in spatial organization, as observed by confocal laser scanning microscopy (CLSM), viable cell content, biovolume, and substratum surface coverage of the biofilms formed on glass by Pseudomonas fluorescens resulting from co-culture with Listeria monocytogenes, were examined. Two strains of L. monocytogenes, two culture temperatures and two biofilm developmental stages were investigated. Both L. monocytogenes strains, a persistently sampled isolate (collected repeatedly along 3 years from a meat factory) and Scott A, induced shrinkage in matrix volume, both at 20°C and 4°C, in mature or old biofilms, without loss of P. fluorescens cell count per surface unit. The nearly homogeneous pattern of surface coverage shown by mono-species P. fluorescens biofilms, turned into more irregular layouts in co-culture with L. monocytogenes. The upper layer of both mono and dual-species biofilms turned to predominantly consist of matrix, with plenty of viable cells underneath, in old biofilms cultured at 20°C, but not in those grown at 4°C. Between 15 and 56% of the substratum area was covered by biofilm, the extent depending on temperature, time and L. monocytogenes strain. Real biofilms in food-related surfaces may thus be very heterogeneous regarding their superficial components, i.e., those more accessible to disinfectants. It is therefore a hygienic challenge to choose an adequate agent to disrupt them. PMID:26913024

  10. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics

    PubMed Central

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E.; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the ‘holy grail’ in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  11. Selected Antimicrobial Essential Oils Eradicate Pseudomonas spp. and Staphylococcus aureus Biofilms

    PubMed Central

    Kavanaugh, Nicole L.

    2012-01-01

    Biofilms are difficult to eliminate with standard antimicrobial treatments due to their high antibiotic resistance relative to free-living cells. Here, we show that selected antimicrobial essential oils can eradicate bacteria within biofilms with higher efficiency than certain important antibiotics, making them interesting candidates for the treatment of biofilms. PMID:22467497

  12. Methicillin Resistant Staphylococcus Aureus Biofilm Formation Over A Separated Flow Region Under Steady And Pulsatile Flow Conditions

    NASA Astrophysics Data System (ADS)

    Salek, M. Mehdi; Martinuzzi, Robert

    2012-02-01

    Several researchers have observed that the formation, morphology and susceptibility of bacterial biofilms are affected by the local hydrodynamic condition and, in particular, shear stresses acting on the fluid-biofilm interface. A backwards facing step (BFS) experimental model has been widely utilized as an in vitro model to examine and characterize the effect of flow separation and recirculation zones comparable to those present within various medical devices as well as those observed in vivo. The specific geometry of BFS covers a vide range of flow features observed in physiological or environmental conditions. The hypothesis of this study is that the flow behavior and structures can effectively contribute to the transport and attachment of cells and affecting the morphology of adhered colonies as well as suspended structures (i.e. biofilm streamers). Hence, the formation of the recirculation region occurring within a backward facing step (BFS) under steady and pulsatile conditions as well as three-dimensional flow structures arising close to the side walls are investigated to correlate to biofilms behavior. This hypothesis is investigated using a backward facing step incorporated into a flow cell under steady and pulsatile flow regimes to study the growth of methicillin resistant Staphylococcus aureus (MRSA) UC18 as the study microorganism.

  13. Comparative Transcriptome Analysis of Desulfovibrio Vulgaris Grown in Planktonic Culture and Mature Biofilm on a Steel Surface

    SciTech Connect

    Zhang, Weiwen; Culley, David E.; Nie, Lei; Scholten, Johannes C.

    2007-08-01

    The build-up of biofilms of sulphate -reducing bacteria (SRB) on metals surfaces may lead to severe corrosion of iron. To understand the processes at molecular level, in this study, a whole-genome oligonucleotide microarray was used to examine differential expression patterns between planktonic populations and mature biofilm of model SRB species Desulfovibrio vulgaris. Statistical analysis revealed that 472 genes were differentially expressed (1.5 fold or more with a p value less than 0.025) when comparing biofilm to planktonic cells. Among the differentially expressed genes were several that corresponded to biofilm formation genes identified in many aerobic bacterial biofilms (i.e., Pseudomonas species and Escherichia coli), such as down-regulation of genes encoding flagellin, flagellar motor switch protein and chemotaxis proteins involved in cell motility and induction of genes encoding sugar transferase and glycogen synthase involved in exopolysaccharide biosynthesis. In addition, D. vulgaris biofilm-bound cells exhibited decreased transcription of genes involved in protein synthesis, energy metabolism and sulfate reduction, as well as genes involved in general stress responses. These findings were all consistent with early suggestion that the average physiology of biofilm cells were similar to planktonic cells of stationary phases. Most notably, up-regulation of large number of outer membrane proteins was observed in D. vulgaris biofilm. Although their function is still unknown, the higher expression of these genes in D. vulgaris biofilm could implicate important roles formation and maintenance of multi-cellular consortium on metal surface. The study provided insights into the metabolic networks associated with D. vulgaris biofilm formation and maintenance on an iron surface.

  14. A comparison of Staphylococcus aureus biofilm formation on cobalt-chrome and titanium-alloy spinal implants.

    PubMed

    Patel, Shalin S; Aruni, Wilson; Inceoglu, Serkan; Akpolat, Yusuf T; Botimer, Gary D; Cheng, Wayne K; Danisa, Olumide A

    2016-09-01

    The use of cobalt chrome (CoCr) implants in spinal surgery has become increasingly popular. However, there have been no studies specifically comparing biofilm formation on CoCr with that of titanium-alloy spinal implants. The objective of this study was to compare the difference in propensity for biofilm formation between these two materials, as it specifically relates to spinal rods. Staphylococcus aureus subsp. Aureus (ATCC 6538) were incubated with two different types of spinal rods composed of either CoCr or titanium-alloy. The spinal rods were then subject to a trypsin wash to allow for isolation of the colonized organism and associated biofilms. The associated optical density values (OD) from the bacterial isolates were obtained and the bacterial solutions were plated on brain-heart infusion agar plates and the resultant colony-forming units (CFU) were counted. The OD values for the titanium-alloy rods were 1.105±0.096nm (mean±SD) and 1.040±0.026nm at 48hours and 96hours, respectively. In contrast, the OD values for the CoCr rods were 1.332±0.161nm and 1.115±0.207nm at 48 and 96hours, respectively (p<0.05). The CFU values were 1481±417/100mm(2) and 745±159/100mm(2) at 48 and 96hours, respectively for the titanium-alloy group. These values were significantly lower than the CFU values obtained from the CoCr group which were 2721±605/100mm(2) and 928±88/100mm(2) (p<0.001) at both 48 and 96hours respectively. Our findings, evaluating both the OD and CFU values, indicate that implants composed of CoCr had a higher proclivity towards biofilm formation compared to titanium-alloy implants. PMID:27396378

  15. Identification of ypqP as a New Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities.

    PubMed

    Sanchez-Vizuete, Pilar; Le Coq, Dominique; Bridier, Arnaud; Herry, Jean-Marie; Aymerich, Stéphane; Briandet, Romain

    2015-01-01

    In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities. PMID:25326298

  16. Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

    PubMed Central

    Sanchez-Vizuete, Pilar; Le Coq, Dominique; Bridier, Arnaud; Herry, Jean-Marie; Aymerich, Stéphane

    2014-01-01

    In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species (“public goods”), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities. PMID:25326298

  17. The role of the globin-coupled sensor YddV in a mature E. coli biofilm population.

    PubMed

    Donné, Joke; Van Kerckhoven, Marian; Maes, Louis; Cos, Paul; Dewilde, Sylvia

    2016-07-01

    Biofilm-associated infections are hard to treat because of their high antibiotic resistance and the presence of a very persistent subpopulation of bacteria. The second messenger molecule cyclic di-guanosine monophosphate (c-di-GMP) plays a very important role in this biofilm physiology. Here, we evaluated the role of YddV, an enzyme with a c-di-GMP synthesis function, in the formation and maturation of Escherichia coli biofilms. Our results suggest that YddV stimulates biofilm growth via its role in the production of c-di-GMP and this likely by influencing the production of matrix (e.g. poly-N-acetylglucosamine (PGA)). However, lowering the YddV expression did not alter the biofilm formation since there was no significant difference between the biofilm phenotypes of WT E. coli and YddV-knockout bacteria. Additionally, YddV expression had no significant influence on the amount of persister cells within the biofilm population, questioning the use of YddV as therapeutic target. PMID:27083533

  18. Evaluation of humoral immunity and protective efficacy of biofilm producing Staphylococcus aureus bacterin-toxoid prepared from a bovine mastitis isolate in rabbit

    PubMed Central

    A., Raza; G., Muhammad; S. U., Rahman; I., Rashid; K., Hanif; A., Atta; S., Sharif

    2015-01-01

    Mastitis is a one of the major diseases of dairy animals. Staphylococcus aureus is the most common microorganism associated with this dairy scourge. Cure rates of mastitis associated with this pathogen are appallingly low. Biofilm is an important virulence factor and immunogenic structure of S. aureus that makes it resistant to phagocytosis and antibiotics. Reports on the efficacy of vaccine prepared from a biofilm producing S. aureus are infrequent. The present study was designed to evaluate the role of a bacterin-toxoid prepared from a strong biofilm producing S. aureus in effective immunization of rabbits. The strong biofilm producing S. aureus selected from 64 isolates of staphylococci was used to prepare bacterin-toxoid and aluminum hydroxide gel was added as an adjuvant. The vaccine was evaluated in rabbits by challenge protection assay and humoral immune response. The mortality rates in control and vaccinated groups were 80% and 10% at day 7 post challenge and 100% and 20% at day 15 post challenge, respectively. Serum antibody titer (GMT) was significantly higher (294.0) in vaccinated group as compared to control group of rabbits (2.63) at day 45. The results showed that the vaccine has significantly elicited humoral immune response in rabbit and developed protective efficacy against new infections. PMID:27175154

  19. Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

    PubMed

    Shakibaie, Mojtaba; Forootanfar, Hamid; Golkari, Yaser; Mohammadi-Khorsand, Tayebe; Shakibaie, Mohammad Reza

    2015-01-01

    The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80-220nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO2 was also evaluated. PMID:25175509

  20. The DUF59 Containing Protein SufT Is Involved in the Maturation of Iron-Sulfur (FeS) Proteins during Conditions of High FeS Cofactor Demand in Staphylococcus aureus.

    PubMed

    Mashruwala, Ameya A; Bhatt, Shiven; Poudel, Saroj; Boyd, Eric S; Boyd, Jeffrey M

    2016-08-01

    Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins. PMID:27517714

  1. The DUF59 Containing Protein SufT Is Involved in the Maturation of Iron-Sulfur (FeS) Proteins during Conditions of High FeS Cofactor Demand in Staphylococcus aureus

    PubMed Central

    Bhatt, Shiven; Poudel, Saroj; Boyd, Eric S.; Boyd, Jeffrey M.

    2016-01-01

    Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins. PMID:27517714

  2. Prevalence of Panton-Valentine leucocidin and phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus strains isolated from children with adenoid hypertrophy.

    PubMed

    Emaneini, Mohammad; Khoramrooz, Seyed Sajjad; Shahsavan, Shadi; Dabiri, Hossein; Jabalameli, Fereshteh

    2015-12-01

    Adenoids as a first line of host defense against respiratory microbes play an important role in majority of upper airway infectious and noninfectious illnesses. Bacterial pathogen can colonize on the adenoid tissue and probably act as a reservoir for them. To determine phenotypic and genotypic characterization of biofilm forming capacity of Staphylococcus aureus isolates from children with adenoid hypertrophy and prevalence of Panton-Valentine leukocidin (PVL) gene we collected 17 consecutive, clinically significant S. aureus isolates from children with adenoid hypertrophy undergoing adenoidectomy with one or more of the upper airway obstruction symptoms, nasal obstruction, mouth breathing, snoring, or sleep apnea. Biofilm formation was evaluated by colorimetric microtiter plate's assay. Gene encoding PVL and adhesion- or biofilm formation-encoding genes were targeted by polymerase chain reaction (PCR) assay. According to the results, all strains produced biofilm. Seven (41.2%) isolates produced strong biofilm whereas 7 (41.2%) isolates produced week and 3 (17.6%) isolates produced medium biofilm. Regarding the adhesion- or biofilm formation-encoding genes, 16 (94.1%) isolates were positive for the gene eno, 13(76.4%) for icaA, 13 (76.4%) for icaD, 10 (58.8%) for fib, 10 (58.8%) for fnbB, 4(23.5%) for can, and 1(5.8%) for fnbA. The high prevalence of genes encoding biofilms and adhesins and phenotypic ability to form a biofilm by S. aureus strains emphasizes the pathogenic character of strains isolated from children with adenoid hypertrophy. PMID:26476288

  3. Interfacial separation of a mature biofilm from a glass surface - A combined experimental and cohesive zone modelling approach.

    PubMed

    Safari, Ashkan; Tukovic, Zeljko; Cardiff, Philip; Walter, Maik; Casey, Eoin; Ivankovic, Alojz

    2016-02-01

    A good understanding of the mechanical stability of biofilms is essential for biofouling management, particularly when mechanical forces are used. Previous biofilm studies lack a damage-based theoretical model to describe the biofilm separation from a surface. The purpose of the current study was to investigate the interfacial separation of a mature biofilm from a rigid glass substrate using a combined experimental and numerical modelling approach. In the current work, the biofilm-glass interfacial separation process was investigated under tensile and shear stresses at the macroscale level, known as modes I and II failure mechanisms respectively. The numerical simulations were performed using a Finite Volume (FV)-based simulation package (OpenFOAM®) to predict the separation initiation using the cohesive zone model (CZM). Atomic force microscopy (AFM)-based retraction curve was used to obtain the separation properties between the biofilm and glass colloid at microscale level, where the CZM parameters were estimated using the Johnson-Kendall-Roberts (JKR) model. In this study CZM is introduced as a reliable method for the investigation of interfacial separation between a biofilm and rigid substrate, in which a high local stress at the interface edge acts as an ultimate stress at the crack tip.This study demonstrated that the total interfacial failure energy measured at the macroscale, was significantly higher than the pure interfacial separation energy obtained by AFM at the microscale, indicating a highly ductile deformation behaviour within the bulk biofilm matrix. The results of this study can significantly contribute to the understanding of biofilm detachments. PMID:26474034

  4. Femtosecond laser surface texturing of titanium as a method to reduce the adhesion of Staphylococcus aureus and biofilm formation

    NASA Astrophysics Data System (ADS)

    Cunha, Alexandre; Elie, Anne-Marie; Plawinski, Laurent; Serro, Ana Paula; Botelho do Rego, Ana Maria; Almeida, Amélia; Urdaci, Maria C.; Durrieu, Marie-Christine; Vilar, Rui

    2016-01-01

    The aim of the present work was to investigate the possibility of using femtosecond laser surface texturing as a method to reduce the colonization of Grade 2 Titanium alloy surfaces by Staphylococcus aureus and the subsequent formation of biofilm. The laser treatments were carried out with a Yb:KYW chirped-pulse-regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. Two types of surface textures, consisting of laser-induced periodic surface structures (LIPSS) and nanopillars, were produced. The topography, chemical composition and phase constitution of these surfaces were investigated by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction. Surface wettability was assessed by the sessile drop method using water and diiodomethane as testing liquids. The response of S. aureus put into contact with the laser treated surfaces in controlled conditions was investigated by epifluorescence microscopy and scanning electron microscopy 48 h after cell seeding. The results achieved show that the laser treatment reduces significantly the bacterial adhesion to the surface as well as biofilm formation as compared to a reference polished surfaces and suggest that femtosecond laser texturing is a simple and promising method for endowing dental and orthopedic titanium implants with antibacterial properties, reducing the risk of implant-associated infections without requiring immobilized antibacterial substances, nanoparticles or coatings.

  5. [Comparison of tigecycline and vancomycin activities in an in vitro biofilm model generated with methicillin-resistant Staphylococcus aureus].

    PubMed

    Aslan, Halil; Yapar, Nur

    2015-10-01

    Today, the most common cause of bloodstream infections, which led to high mortality, prolonged hospitalization and increased costs are the intravenous catheters. Among the microorganisms associated with catheter infections, staphylococci took the first place and because of their biofilm-forming properties they cause serious problems in treatment and management of the patients. Although the drug of choice in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection is vancomycin, its effect on the bacterial biofilm is known to be low. Tigecycline, newly used in our country is a well tolerated glycylcycline antibiotic. In this study, we aimed to compare the efficacy of tigecycline and vancomycin in an in vitro MRSA biofilm model. The study consisted of 10 MRSA strains, which were detected as causative agents of catheter-related infections in our hospital. The methicillin resistance of the strains were performed by disk diffusion test with oxacillin (1 μg) disks and the biofilm forming capacity of the strains was evaluated using the Congo red agar method. The silicone disks with created biofilm layer were exposed to tigecycline (2 mg/ml) and vancomycin (2 mg/ml) for 24 hours and for 5 days 4-hours per day in a model of antibiotic lock therapy. The present study showed that, after incubating the silicon discs in antibiotic solution for 24 hours, colony forming unit counts of MRSA decreased from 10(5) cfu/ml to 510 cfu/ml in the tigecycline group and from 105 cfu/ml to 3.800 cfu/ml in the vancomycin group and remained the same in the control (10(5) cfu/ml) group (p< 0.001). In the antibiotic lock therapy model, incubation with antibiotics for 4 hours per day, yielded that the average growth was 1.800 cfu/ml in the tigecycline group and 8.700 cfu/ml in the vancomycin group, which was statistically significant (p< 0.001). No growth was detected in the tigecycline group (0 cfu/ml) while in vancomycin group number of colonies in second, thirth and

  6. Nisin Incorporated With 2,3-Dihydroxybenzoic Acid in Nanofibers Inhibits Biofilm Formation by a Methicillin-Resistant Strain of Staphylococcus aureus.

    PubMed

    Ahire, Jayesh J; Dicks, Leon M T

    2015-03-01

    The aim of the present study was to determine the effect of nisin, 2,3-dihydroxybenzoic acid (DHBA) and a combination of nisin and DHBA incorporated into nanofibers prepared from poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO) on biofilm formation of a methicillin-resistant strain of Staphylococcus aureus (strain Xen 31). Biofilm formation decreased by 88% after 24 h of exposure to nanofibers containing nisin and DHBA (NDF), compared to a 63% decrease when exposed to nanofibers containing only DHBA (DF) and a 3% decrease when exposed to nanofibers containing only nisin (NF). Planktonic cell numbers of biofilms exposed to nanofibers without nisin or DHBA (CF) and NF increased from no detectable OD(595nm) readings to 0.35 and 0.3, respectively, within the first 8 h of exposure, followed by a steady decline over the following 16 h. Planktonic cells of biofilms treated with DF increased from no detectable OD(595nm) readings to 0.05 after 8 h of exposure and remained more-or-less constant for the duration of the experiment. Planktonic cells of biofilms exposed to NDF increased from OD(595nm) 0.03 after 8 h of exposure and to 0.2 over the following 16 h. Biofilm formation increased with increasing concentrations of FeCl3·6H2O, which suggests that iron is required for S. aureus Xen 31 to form a biofilm. However, when exposed to NDF, biofilm formation decreased significantly in the presence of increasing concentrations of iron. This suggests that NDF may be used to prevent biofilm formation of MRSA and control infection. PMID:25319566

  7. Impact of Vancomycin on sarA-Mediated Biofilm Formation: Role in Persistent Endovascular Infections Due to Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Abdelhady, Wessam; Bayer, Arnold S.; Seidl, Kati; Moormeier, Derek E.; Bayles, Kenneth W.; Cheung, Ambrose; Yeaman, Michael R.; Xiong, Yan Q.

    2014-01-01

    Background. Staphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation. Methods. Two healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub–minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model. Results. All sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model. Conclusions. These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation. PMID:24403556

  8. Role of initial contamination levels, biofilm maturity and presence of salt and fat on desiccation survival of Listeria monocytogenes on stainless steel surfaces.

    PubMed

    Hingston, Patricia A; Stea, Emma C; Knøchel, Susanne; Hansen, Truelstrup

    2013-10-01

    This study investigated the effect of initial contamination levels, biofilm maturity and presence of salt and fatty food soils on desiccation survival of Listeria monocytogenes on stainless steel (SS) coupons. L. monocytogenes cultures grown (at 15 °C for 48 h) in Tryptic Soy Broth with 1% glucose (TSB-glu) containing either 0.5 or 5% (w/v) NaCl were re-suspended in TSB-glu containing either 0.5 or 5% NaCl and used to contaminate SS coupons at levels of 3.5, 5.5, and 7.5 log CFU/cm². Desiccation (at 15 °C for 20 days, 43% RH) commenced immediately (non-biofilm) or following biofilm formation (at 15 °C for 48 h, 100% RH). To study the impact of food lipids, non-biofilm L. monocytogenes cells were suspended in TSB-glu containing either canola oil (5-10%) or lard (20-60%) and desiccated as above on SS coupons. Following desiccation for 20 days, survivors decreased by 1.4-3.7 log CFU/cm² for non-biofilm L. monocytogenes cells. The contamination level had no significant (p > 0.05) effect on survival kinetics. SEM micrographs showed mature biofilms on coupons initially contaminated with 5.5 and 7.5 log CFU/cm². Mature biofilm cells were significantly (p < 0.05) more desiccation resistant than cells in immature biofilms formed by the lowest contamination level. Besides biofilm maturity/formation, previous osmoadaptation, exposure to lard (20-60%) or salt (5%) during desiccation significantly (p < 0.05) increased the bacterium's survival. In conclusion, L. monocytogenes desiccation survival can be greatly reduced by preventing presence of mature biofilms and salty or fatty soils on food contact surfaces. PMID:23764219

  9. Mature Biofilm Degradation by Potential Probiotics: Aggregatibacter actinomycetemcomitans versus Lactobacillus spp.

    PubMed Central

    Mizuno, Kouhei; Okinaga, Toshinori

    2016-01-01

    The biofilm degradation of Aggregatibacter actinomycetemcomitans is essential as a complete periodontal disease therapy, and here we show the effects of potential probiotic bacteria such as Lactobacillus spp. for the biofilm of several serotypes of A. actinomycetemcomitans strains. Eight of the 13 species showed the competent biofilm degradation of ≥ 90% reduction in biofilm values in A. actinomycetemcomitans Y4 (serotype b) as well as four of the seven species for the biofilm of A. actinomycetemcomitans OMZ 534 (serotype e). In contrast, the probiotic bacteria did not have a big impact for the degradation of A. actinomycetemcomitans SUNY 75 (serotype a) biofilm. The dispersed A. actinomycetemcomitans Y4 cells through the biofilm detachment were still viable and plausible factors for the biofilm degradation were not due to the lactic acid and low pH conditions. The three enzymes, protease, lipase, and amylase may be responsible for the biofilm degradation; in particular, lipase was the most effective enzyme for the biofilm degradation of A. actinomycetemcomitans Y4 along with the protease activity which should be also important for the other serotypes. Remarkable lipase enzyme activities were detected from some of the potential probiotics and a supporting result using a lipase inhibitor presented corroborating evidence that lipase activity is one of the contributing factors for biofilm degradation outside of the protease which is also another possible factor for the biofilm of the other serotype of A. actinomycetemcomitans strains. On the other hand, the biofilm of A. actinomycetemcomitans SUNY 75 (serotype a) was not powerfully degraded by the lipase enzyme because the lipase inhibitor was slightly functional for only two of potential probiotics. PMID:27438340

  10. Impact of the Maturation of Human Primary Bone-Forming Cells on Their Behavior in Acute or Persistent Staphylococcus aureus Infection Models

    PubMed Central

    Josse, Jérôme; Guillaume, Christine; Bour, Camille; Lemaire, Flora; Mongaret, Céline; Draux, Florence; Velard, Frédéric; Gangloff, Sophie C.

    2016-01-01

    Staphylococcus aureus is one of the most frequently involved pathogens in bacterial infections such as skin abscess, pneumonia, endocarditis, osteomyelitis, and implant-associated infection. As for bone homeostasis, it is partly altered during infections by S. aureus by the induction of various responses from osteoblasts, which are the bone-forming cells responsible for extracellular matrix synthesis and its mineralization. Nevertheless, bone-forming cells are a heterogeneous population with different stages of maturation and the impact of the latter on their responses toward bacteria remains unclear. We describe the impact of S. aureus on two populations of human primary bone-forming cells (HPBCs) which have distinct maturation characteristics in both acute and persistent models of interaction. Cell maturation did not influence the internalization and survival of S. aureus inside bone-forming cells or the cell death related to the infection. By studying the expression of chemokines, cytokines, and osteoclastogenic regulators by HPBCs, we observed different profiles of chemokine expression according to the degree of cell maturation. However, there was no statistical difference in the amounts of proteins released by both populations in the presence of S. aureus compared to the non-infected counterparts. Our findings show that cell maturation does not impact the behavior of HPBCs infected with S. aureus and suggest that the role of bone-forming cells may not be pivotal for the inflammatory response in osteomyelitis. PMID:27446812

  11. Impact of the Maturation of Human Primary Bone-Forming Cells on Their Behavior in Acute or Persistent Staphylococcus aureus Infection Models.

    PubMed

    Josse, Jérôme; Guillaume, Christine; Bour, Camille; Lemaire, Flora; Mongaret, Céline; Draux, Florence; Velard, Frédéric; Gangloff, Sophie C

    2016-01-01

    Staphylococcus aureus is one of the most frequently involved pathogens in bacterial infections such as skin abscess, pneumonia, endocarditis, osteomyelitis, and implant-associated infection. As for bone homeostasis, it is partly altered during infections by S. aureus by the induction of various responses from osteoblasts, which are the bone-forming cells responsible for extracellular matrix synthesis and its mineralization. Nevertheless, bone-forming cells are a heterogeneous population with different stages of maturation and the impact of the latter on their responses toward bacteria remains unclear. We describe the impact of S. aureus on two populations of human primary bone-forming cells (HPBCs) which have distinct maturation characteristics in both acute and persistent models of interaction. Cell maturation did not influence the internalization and survival of S. aureus inside bone-forming cells or the cell death related to the infection. By studying the expression of chemokines, cytokines, and osteoclastogenic regulators by HPBCs, we observed different profiles of chemokine expression according to the degree of cell maturation. However, there was no statistical difference in the amounts of proteins released by both populations in the presence of S. aureus compared to the non-infected counterparts. Our findings show that cell maturation does not impact the behavior of HPBCs infected with S. aureus and suggest that the role of bone-forming cells may not be pivotal for the inflammatory response in osteomyelitis. PMID:27446812

  12. Effect of essential oils of Syzygium aromaticum and Cinnamomum zeylanicum and their major components on biofilm production in Staphylococcus aureus strains isolated from milk of cows with mastitis.

    PubMed

    Budri, P E; Silva, N C C; Bonsaglia, E C R; Fernandes Júnior, A; Araújo Júnior, J P; Doyama, J T; Gonçalves, J L; Santos, M V; Fitzgerald-Hughes, D; Rall, V L M

    2015-09-01

    Bovine mastitis is an inflammation of the mammary glands of cows and causes significant economic losses in dairy cattle. Staphylococcus aureus is one of the microorganisms most commonly isolated. Novel agents are required in agricultural industries to prevent the development of mastitis. The production of biofilm by Staph. aureus facilitates the adhesion of bacteria to solid surfaces and contributes to the transmission and maintenance of these bacteria. The effect of the essential oils of Syzygium aromaticum (clove; EOSA) and Cinnamomum zeylanicum (cinnamon; EOCZ) and their major components, eugenol and cinnamaldehyde, on Staph. aureus biofilm formation on different surfaces was investigated. The results showed a significant inhibition of biofilm production by EOSA on polystyrene and stainless steel surfaces (69.4 and 63.6%, respectively). However, its major component, eugenol, was less effective on polystyrene and stainless steel (52.8 and 19.6%, respectively). Both EOCZ and its major component, cinnamaldehyde, significantly reduced biofilm formation on polystyrene (74.7 and 69.6%, respectively) and on stainless steel surfaces (45.3 and 44.9%, respectively). These findings suggest that EOSA, EOCZ, and cinnamaldehyde may be considered for applications such as sanitization in the food industry. PMID:26142866

  13. In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [18F]fluoro-deoxyglucose-MicroPET in a mouse model.

    PubMed

    Garrido, Victoria; Collantes, María; Barberán, Montserrat; Peñuelas, Iván; Arbizu, Javier; Amorena, Beatriz; Grilló, María-Jesús

    2014-11-01

    A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [(18)F]fluoro-deoxyglucose-MicroPET ([(18)F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [(18)F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [(18)F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches. PMID:25155589

  14. In Vivo Monitoring of Staphylococcus aureus Biofilm Infections and Antimicrobial Therapy by [18F]Fluoro-Deoxyglucose–MicroPET in a Mouse Model

    PubMed Central

    Garrido, Victoria; Collantes, María; Barberán, Montserrat; Peñuelas, Iván; Arbizu, Javier; Amorena, Beatriz

    2014-01-01

    A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [18F]fluoro-deoxyglucose–MicroPET ([18F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [18F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [18F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches. PMID:25155589

  15. In vitro effectiveness of 455-nm blue LED to reduce the load of Staphylococcus aureus and Candida albicans biofilms in compact bone tissue.

    PubMed

    Rosa, Luciano Pereira; da Silva, Francine Cristina; Viana, Magda Souza; Meira, Giselle Andrade

    2016-01-01

    The aim of this study was to evaluate the effectiveness of a 455-nm blue light-emitting diode (LED), at different application times, to reduce the load of Staphylococcus aureus and Candida albicans biofilms applied to compact bone tissue. The microorganisms S. aureus (ATCC 25923) and C. albicans (ATCC 18804) were used to form biofilms on 160 specimens of compact bones that had been divided into eight experimental groups (n = 10) for each microorganism, according to the times of application of the 455-nm blue LED (1, 2, 3, 4, 5, 7, and 10 min) with an irradiance of 75 mW/cm2. After LED application, decimal dilutions of microorganisms were performed, plated on BHI or Sabouraud agar and incubated for 24 h/35 °C to obtain CFU/mL counts. The findings were statistically analyzed using a ANOVA 5 %. For the group of S. aureus biofilms, all groups of 455-nm LED application differ compared with the control group (p < 0.05), in which no treatment was given. The largest reduction was obtained in the group receiving LED for 10 min (p = 0.00); within this group, a 3.2 log reduction was observed. For the C. albicans biofilms, only those samples receiving 3, 7, and 10 min of LED application presented a significant difference compared with the control group (p < 0.00), indicating that longer application times are required to achieve efficacy. The results of this study show that 455-nm LED light was effective to reduce the load of S. aureus and C. albicans biofilms, especially during 10 min of application. PMID:26498601

  16. Biofilms

    PubMed Central

    van Hoek, Monique L

    2013-01-01

    Our understanding of the virulence and pathogenesis of Francisella spp. has significantly advanced in recent years, including a new understanding that this organism can form biofilms. What is known so far about Francisella spp. biofilms is summarized here and future research questions are suggested. The molecular basis of biofilm production has begun to be studied, especially the role of extracellular carbohydrates and capsule, quorum sensing and two-component signaling systems. Further work has explored the contribution of amoebae, pili, outer-membrane vesicles, chitinases, and small molecules such as c-di-GMP to Francisella spp. biofilm formation. A role for Francisella spp. biofilm in feeding mosquito larvae has been suggested. As no strong role in virulence has been found yet, Francisella spp. biofilm formation is most likely a key mechanism for environmental survival and persistence. The significance and importance of Francisella spp.’s biofilm phenotype as a critical aspect of its microbial physiology is being developed. Areas for further studies include the potential role of Francisella spp. biofilms in the infection of mammalian hosts and virulence regulation. PMID:24225421

  17. Comparative Efficacies of Tedizolid Phosphate, Linezolid, and Vancomycin in a Murine Model of Subcutaneous Catheter-Related Biofilm Infection Due to Methicillin-Susceptible and -Resistant Staphylococcus aureus.

    PubMed

    Bayer, Arnold S; Abdelhady, Wessam; Li, Liang; Gonzales, Rachelle; Xiong, Yan Q

    2016-08-01

    Tedizolid, a novel oxazolidinone, exhibits bacteriostatic activity through inhibition of protein synthesis. The efficacies of tedizolid, linezolid, and vancomycin were compared in a murine catheter-related biofilm infection caused by methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) strains engineered for bioluminescence. We observed significantly improved efficacy in terms of decreased S. aureus densities and bioluminescent signals in the tedizolid-treated group versus the linezolid- and vancomycin-treated groups in the model of infection caused by the MSSA and MRSA strains. PMID:27297485

  18. Correlation of mupirocin resistance with biofilm production in methicillin-resistant Staphylococcus aureus from surgical site infections in a tertiary centre, Egypt.

    PubMed

    Barakat, Ghada I; Nabil, Yasmin M

    2016-03-01

    The aim of this study was to detect mupirocin-resistant isolates from pus/wound swabs taken postoperatively in a tertiary centre in Egypt and to determine their ability to form biofilm in order to establish its correlation with mupirocin resistance. This was a prospective study including 513pus/wound swabs from patients suffering from postoperative surgical site infections over the period July 2013-January 2015. Samples were cultured and isolates were identified by coagulase activity, DNase test, mannitol fermentation by mannitol salt agar followed by API Staph 32. Oxacillin agar screen test, agar dilution test for mupirocin, and mupA gene detection by PCR were performed for all methicillin-resistant Staphylococcus aureus (MRSA) isolates. Biofilm detection was carried out by the microtitre plate and Congo red agar methods. Of the 161 S. aureus isolates identified, 73 (45.3%) were MRSA, among which 82.2% were mupirocin-susceptible and 17.8% were mupirocin-resistant. Among the resistant isolates, 38.5% showed low-level resistance and 61.5% were high-level mupirocin-resistant. The mupA gene was detected in 75.0% of high-level mupirocin-resistant strains and in none of the low-level mupirocin-resistant strains. Among the mupirocin-susceptible isolates, 95.0% were biofilm-producers and 5.0% did not produce biofilm. All mupirocin-resistant isolates produced biofilm. Moreover, 15.3% of high-level mupirocin-resistant strains were negative for the mupA gene but showed evidence of biofilm formation. In conclusion, biofilm formation may be suggested to play a role in mupirocin resistance besides the presence of a genetic element encoding abnormal isoleucyl-tRNA synthetase, however further studies are needed to confirm these findings. PMID:27436387

  19. Proteome Analyses of Staphylococcus aureus Biofilm at Elevated Levels of NaCl

    PubMed Central

    Islam, Nazrul; Ross, Julia M; Marten, Mark R

    2016-01-01

    Our studies demonstrate that sodium chloride (NaCl) induces changes in biofilm, mediated by increased production of polysaccharides intercellular adhesion (PIA). We identified 12 proteins that showed higher abundance in increased level of NaCl. This includes one important protein (IsaA) known to be associated with biofilm stability. In addition, we also found higher abundance of a cold shock protein, CspA, at higher NaCl. We have also identified several other proteins that are differentially expressed to the elevated levels of NaCl and mapped them in the regulatory pathways of PIA. The majority of proteins are involved with various aspects bacterial metabolic function. Our results demonstrated that NaCl influences gene regulatory networks controlling exopolysaccharide expression. PMID:26973848

  20. Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination.

    PubMed

    Flannagan, Ronald S; Heit, Bryan; Heinrichs, David E

    2016-04-01

    The success of Staphylococcus aureus as a pathogen is partly attributable to its ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of phagocytes. Here, we have studied the interaction of methicillin-resistant S. aureus (MRSA) strain USA300 with murine RAW 264.7 and primary human macrophages using molecular imaging and single cell analysis to obtain an unprecedented understanding of the interaction between the macrophage and MRSA. Herein we demonstrate that macrophages fail to control intracellular infection by MRSA USA300 despite trafficking the bacteria into mature phagolysosomes. Using fluorescence-based proliferation assays we also show that intracellular staphylococci proliferate and that replication commences while the bacteria are residing in mature phagolysosomes hours after initial phagocytosis. Finally, live-cell fluorescence video microscopy allowed for unprecedented visual insight into the escape of MRSA from macrophages, demonstrating that the macrophages die through a pathway characterized by membrane blebbing and activation of caspase-3 followed by acquisition of the vital dye propidium iodide. Moreover, cell death precedes the emergence of MRSA from infected macrophages, and these events can be ablated by prolonged exposure of infected phagocytes to gentamicin. PMID:26408990

  1. Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae

    NASA Astrophysics Data System (ADS)

    Vijayakumar, S.; Vinoj, G.; Malaikozhundan, B.; Shanthi, S.; Vaseeharan, B.

    2015-02-01

    In this study, zinc oxide nanoparticles were biologically synthesized using the leaf extract of Plectranthus amboinicus (Pam-ZnO NPs). The synthesized Pam-ZnO NPs were characterized by UV-Vis spectrophotometer, FTIR, TEM and XRD analysis. TEM analysis of Pam-ZnO NPs showed the average size of about 20-50 nm. Pam-ZnO NPs control the growth of methicillin-resistant Staphylococcus aureus biofilms (MRSA ATCC 33591) at the concentration of 8-10 μg/ml. Confocal laser scanning microscope (CLSM) images revealed that Pam-ZnO NPs strongly inhibited the biofilm forming ability of S. aureus. In addition, Pam-ZnO NPs showed 100% mortality of fourth instar mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Culex tritaeniorhynchus at the concentration of 8 and 10 μg/ml. The histopathological studies of Pam-ZnO NPs treated A. stephensi and C. quinquefasciatus larvae revealed the presence of damaged cells and tissues in the mid-gut. The damaged tissues suffered major changes including rupture and disintegration of epithelial layer and cellular vacuolization. The present study conclude that Pam-ZnO NPs showed effective control of S. aureus biofilms and mosquito larvae by damaging the mid gut cells.

  2. Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae.

    PubMed

    Vijayakumar, S; Vinoj, G; Malaikozhundan, B; Shanthi, S; Vaseeharan, B

    2015-02-25

    In this study, zinc oxide nanoparticles were biologically synthesized using the leaf extract of Plectranthus amboinicus (Pam-ZnO NPs). The synthesized Pam-ZnO NPs were characterized by UV-Vis spectrophotometer, FTIR, TEM and XRD analysis. TEM analysis of Pam-ZnO NPs showed the average size of about 20-50 nm. Pam-ZnO NPs control the growth of methicillin-resistant Staphylococcus aureus biofilms (MRSA ATCC 33591) at the concentration of 8-10 μg/ml. Confocal laser scanning microscope (CLSM) images revealed that Pam-ZnO NPs strongly inhibited the biofilm forming ability of S. aureus. In addition, Pam-ZnO NPs showed 100% mortality of fourth instar mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Culex tritaeniorhynchus at the concentration of 8 and 10 μg/ml. The histopathological studies of Pam-ZnO NPs treated A. stephensi and C. quinquefasciatus larvae revealed the presence of damaged cells and tissues in the mid-gut. The damaged tissues suffered major changes including rupture and disintegration of epithelial layer and cellular vacuolization. The present study conclude that Pam-ZnO NPs showed effective control of S. aureus biofilms and mosquito larvae by damaging the mid gut cells. PMID:25280336

  3. Increased Effects of Extracorporeal Shock Waves Combined with Gentamicin against Staphylococcus aureus Biofilms In Vitro and In Vivo.

    PubMed

    Qi, Xin; Zhao, Yaochao; Zhang, Jieyuan; Han, Dan; Chen, Chunyuan; Huang, Yinjun; Chen, Xiaofeng; Zhang, Xianlong; Wang, Ting; Li, Xiaolin

    2016-09-01

    An implant-associated bacterial infection is one of the most common and costly complications of orthopedic surgery. Once biofilms develop, it is extremely difficult to cure infections with antimicrobial agents. High-energy extracorporeal shock wave (ESW) treatment has been used for orthopedic-related diseases and has been found to be an effective bactericidal agent that is tolerable both in vitro and in vivo. The broad-spectrum antibiotic gentamicin exhibits bactericidal activity against Staphylococcus aureus, and bacterial resistance to gentamicin is lower. We tested the effectiveness of gentamicin in combination with ESW treatment against S. aureus biofilms in vivo and in vitro. The spread plate method, crystal violet staining, confocal laser scanning microscopy, scanning electron microscopy and microbiologic evaluation were used to compare the effects of combined treatment with those of either treatment alone. The results revealed statistically significant differences between the group treated with ESWs combined with gentamicin and all other groups. Our findings indicate that use of the combination of ESWs with gentamicin is more effective against S. aureus biofilms in vitro and in vivo. PMID:27260244

  4. Analysis of meticillin-susceptible and meticillin-resistant biofilm-forming Staphylococcus aureus from catheter infections isolated in a large Italian hospital.

    PubMed

    Petrelli, Dezemona; Repetto, Antonella; D'Ercole, Stefania; Rombini, Silvia; Ripa, Sandro; Prenna, Manuela; Vitali, Luca Agostino

    2008-03-01

    Several characteristics were analysed in 37 Staphylococcus aureus isolates from nosocomial catheter infections: the PFGE profile after SmaI digestion of chromosomal DNA, the ability to form a biofilm on a polystyrene surface, antibiotic susceptibility patterns (penicillin, oxacillin, erythromycin, tetracycline, clindamycin, telithromycin, gentamicin, ciprofloxacin, quinupristin/dalfopristin, rifampicin, vancomycin and linezolid), and the presence of genetic determinants of antibiotic resistance and biofilm formation. All strains but three (92 %) were able to grow on a plastic surface as a biofilm. An almost complete association was found between phenotypes and genotypic traits of antibiotic resistance, whilst PFGE profiling showed the highly polyclonal composition of the set of strains under study. Sixteen isolates (43 %) were meticillin-resistant and were subjected to staphylococcal cassette chromosome mec (SCCmec) and cassette chromosome recombinase (ccr) complex type determination by multiplex PCR. Only a subgroup of six strains belonged to the archaic clone PFGE type and bore the SCCmec/ccrAB type I structure. Among the remaining strains some presented small rearrangements of the SCCmec/ccrAB genetic locus, whilst others could barely be traced back to a known structural type. These observations suggest that, at the local level and at a particular site of infection, S. aureus may show great genetic variability and escape the general rule of expansion of the S. aureus pandemic clones. PMID:18287301

  5. Rifampicin-containing combinations are superior to combinations of vancomycin, linezolid and daptomycin against Staphylococcus aureus biofilm infection in vivo and in vitro.

    PubMed

    Jørgensen, Nis Pedersen; Skovdal, Sandra M; Meyer, Rikke L; Dagnæs-Hansen, Frederik; Fuursted, Kurt; Petersen, Eskild

    2016-06-01

    Susceptibility to antibiotics is dramatically reduced when bacteria form biofilms. In clinical settings this has a profound impact on treatment of implant-associated infections, as these are characterized by biofilm formation. Current routine susceptibility testing of microorganisms from infected implants does not reflect the actual susceptibility, and the optimal antibiotic strategy for treating implant-associated infections is not established. In this study of biofilm formation in implant-associated osteomyelitis, we compared thein vitroandin vivoefficacy of selected antibiotics alone and in combination againstStaphylococcus aureus.We tested vancomycin, linezolid, daptomycin and tigecycline alone and in combination with rifampicin, vancomycin, linezolid and daptomycin againstS. aureusIn vitro, biofilm formation dramatically reduced susceptibility by a factor of 500-2000.In vivo, antibiotic combinations were tested in a murine model of implant-associated osteomyelitis. Mice were infected by inserting implants colonized withS. aureustrough their tibia. After 11 days, the animals were divided into different groups (five animals/group) and given 14 days of antibiotic therapy. All antibiotics resulted in a reduced bacterial load in the infected bone surrounding the implant. Overall, the most effective antibiotic combinations contained rifampicin. Combinations containing two non-rifampicin antibiotics were not more active than single drugs. PMID:27036412

  6. Chamaecyparis obtusa Essential Oil Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm Formation and Expression of Virulence Factors.

    PubMed

    Kim, Eun-Sook; Kang, Sun-Young; Kim, Young-Hoi; Lee, Young-Eun; Choi, Na-Young; You, Yong-Ouk; Kim, Kang-Ju

    2015-07-01

    The emergence of antibiotic-resistant bacteria has caused difficulty in treating infectious diseases. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most commonly recognized antibiotic-resistant bacteria. Novel antibiotics are urgently required to treat these bacteria. Raw materials derived from natural sources can be used for the development of novel antibiotics, such as Chamaecyparis obtusa (C. obtusa), which has been traditionally used in treating asthmatic disease. In this study, the antibacterial activity of the essential oil (EO) extracted from C. obtusa leaves against MRSA was investigated. MRSA growth and acid production from glucose metabolism were inhibited at concentrations greater than 0.1 mg/mL C. obtusa EO. MRSA biofilm formation was observed using scanning electron microscopy and safranin staining. C. obtusa EO inhibited MRSA biofilm formation at concentrations greater than 0.1 mg/mL. Using real-time polymerase chain reaction, mRNA expression of virulence factor genes, sea, agrA, and sarA, was observed. agrA expression was inhibited with C. obtusa EO concentrations greater than 0.2 mg/mL, whereas inhibition of sea and sarA expression was also observed at a concentration of 0.3 mg/mL. C. obtusa EO was analyzed by gas chromatography (GC) and GC coupled for mass spectrometry, which identified 59 constituents, accounting to 98.99% of the total EO. These findings suggest that C. obtusa EO has antibacterial effects against MRSA, which might be associated with the major components of C. obtusa EO, such as sabinene (19.06%), α-terpinyl acetate (16.99%), bornyl acetate (10.48%), limonene (8.54%), elemol (7.47%), myrcene (5.86%), γ-terpinene (4.04%), and hibaene (3.01%). PMID:25923444

  7. Relationship between multiple drug resistance and biofilm formation in Staphylococcus aureus isolated from medical and non-medical personnel in Yaounde, Cameroon

    PubMed Central

    Eyoh, Agnes Bedie; Toukam, Michel; Atashili, Julius; Fokunang, Charles; Gonsu, Hortense; Lyonga, Emilia Enjema; Mandi, Henshaw; Ikomey, George; Mukwele, Bertha; Mesembe, Martha; Assoumou, Marie Claire Okomo

    2014-01-01

    Introduction Monitoring the prevalence of nasal carriage of multiple drug resistance (MDR) Staphylococcus aureus (SA) strains in hospital personnel is essential. These strains when transmitted from hospital personnel to patients with already weakened immune states or in-built medical devices, may limit the latter's treatment options. This study aimed at assessing the potential exposure of patients to these MDR SA in a resource-limited hospital setting by assessing the prevalence and relationship between antimicrobial susceptibility and biofilm forming capacity of SA isolates from hospital personnel. Methods A total of 59 bacteria isolates phenotypically identified as Staphylococcus aureus obtained from medical (39) and non-medical personnel (20) in Yaounde were used in the study. Multiple drug resistance defined as resistance to four or more of twelve locally used antibiotics were determined by Kirby Bauer disc diffusion technique whereas quantification of biofilm production was by the microtitre plate method. Results Among the 59 SA isolates, the prevalence of MDR was 50.9%. Among medical personnel 48.7% had MDR as against 55.9% for non-medical personnel (p-value=0.648). The overall percentage of weak biofilm producers was 35.6%. Although the prevalence of weak biofilm formers was higher in isolates from non-medical personnel (40%) than medical personnel (33.3%) the difference was not statistically significant (p-value= 0.246). Slightly less than half (42.9%) of the weak biofilm producers were MDR. Conclusion Considering the high rates of MDR and that slightly less than half of biofilm formers were MDR, these trends need to be monitored regularly among hospital personnel in Yaounde. PMID:25396012

  8. Properties of silver and copper nanoparticle-containing aqueous solutions and evaluation of their in vitro activity against Candida albicans and Staphylococcus aureus biofilms

    NASA Astrophysics Data System (ADS)

    Montes Aguirre, Melissa Mariluz

    Most microorganisms grow on surfaces as biofilms rather than as individual planktonic cells, and cells within biofilms show high levels of resistance against antimicrobial drugs. Thereby biofilm formation complicates treatment and contributes to high morbidity and mortality rates associated with infections. This study explores the physical, optical, and nano-structural properties of selected nanoparticles dispersed in aqueous solutions (nanoparticulate colloidal water or nanofluids) and examines their in vitro activity against microbial biofilms. Silver and copper nanofluids of various concentrations were prepared and studied. Their surface energies, surface charge and surface plasmonic resonance properties were obtained using contact angle measurement, zeta potential and optical spectrometer, respectively. The temperature dependence of the surface plasmon resonance behavior was also determined for the selected nanoparticulate aqueous solutions. A model of biofilm formation on the wells of microtiter plates was used to determine the in vitro activity of the nanoparticle preparations against both fungal (Candida albicans) and bacterial (Staphylococcus aureus) biofilms. Scanning electron microscopy (SEM) was used to observe the nanoparticle interactions with microbial cells. Results show that silver nanofluid has higher surface energy than that of the copper, the surface energy increases as the concentration of silver nanoparticles increases; and both nanoparticles in liquid are positively charged. The interaction between silver nanoparticles and water molecules produces notable changes on the usual temperature properties of water. Altogether, effectiveness of silver nanoparticle-containing liquids in controlling biofilm formation is observed and reported. For a given size of silver nanoparticles studied, it is found that the effective concentrations of silver nanoparticles against microbial biofilms are far lower than their cytotoxic concentrations, indicating an

  9. Ethanol extract of Sanguisorba officinalis L. inhibits biofilm formation of methicillin-resistant Staphylococcus aureus in an ica-dependent manner.

    PubMed

    Chen, Xiaolin; Shang, Fei; Meng, Yajing; Li, Long; Cui, Yunmei; Zhang, Ming; Qi, Kezong; Xue, Ting

    2015-12-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen that shows resistance to many antibiotics and is usually associated with serious infections. Having the ability for biofilm formation increases resistance to antibiotics. Sanguisorba officinalis L. is a perennial plant that is distributed in the northern districts of China and has been used as a traditional Chinese medicine. In this study, the effect of S. officinalis on MRSA strain SA3 isolated from a dairy cow with mastitis was evaluated by testing the growth and biofilm formation ability of MRSA cultured with or without ethanol extracts of S. officinalis. The results showed that the ethanol extract of S. officinalis strongly inhibited the biofilm formation of MRSA. With a confocal laser scanning microscope system, we observed that the biofilm structure of the test group with the addition of S. officinalis appeared looser and had less biomass compared with the control group without S. officinalis. Furthermore, we found that the transcript levels of the icaADBC operon remarkably decreased upon addition of the ethanol extract of S. officinalis, indicating that S. officinalis inhibits biofilm formation of MRSA in an ica-dependent manner. PMID:26454299

  10. Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development

    PubMed Central

    Atshan, Salman S.; Shamsudin, Mariana N.; Sekawi, Zamberi; Thian Lung, Leslie T.; Barantalab, Fatemeh; Liew, Yun K.; Alreshidi, Mateg Ali; Abduljaleel, Salwa A.; Hamat, Rukman A.

    2015-01-01

    Staphylococcus aureus is well known for its biofilm formation with rapid emergence of new clones circulating worldwide. The main objectives of the study were (1) to identify possible differences in protein expression among various and closely related clonal types of S. aureus, (2) to establish the differences in protein expression in terms of size of protein spots and its intensities between bacteria which are grown statically (biofilm formation) with that of under aeration and agitation, and (3) to compare the differences in protein expression as a function of time (in hours). In this study, we selected six clinical isolates comprising two similar (MRSA-527 and MRSA-524) and four different (MRSA-139, MSSA-12E, MSSA-22d, and MSSA-10E) types identified by spa typing, MLST and SCCmec typing. We performed 2D gel migration comparison. Also, two MRSA isolates (527 and 139) were selected to determine quantitative changes in the level of extracellular proteins at different biofilm growth time points of 12, 24, and 48 h. The study was done using a strategy that combines 2-DGE and LC-MS/MS analysis for absolute quantification and identification of the extracellular proteins. The 2DGE revealed that the proteomic profiles for the isolates belonging to the similar spa, MLST, and SCCmec types were still quite different. Among the extracellular proteins secreted at different time points of biofilm formation, significant changes in protein expression were observed at 48 h incubation as compared to the exponential growth at 12 h incubation. The main conclusion of the work is that the authors do observe differences among isolates, and growth conditions do influence the protein content at different time points of biofilm formation. PMID:26089817

  11. Influence of Adhesion Force on icaA and cidA Gene Expression and Production of Matrix Components in Staphylococcus aureus Biofilms.

    PubMed

    Harapanahalli, Akshay K; Chen, Yun; Li, Jiuyi; Busscher, Henk J; van der Mei, Henny C

    2015-05-15

    The majority of human infections are caused by biofilms. The biofilm mode of growth enhances the pathogenicity of Staphylococcus spp. considerably, because once they adhere, staphylococci embed themselves in a protective, self-produced matrix of extracellular polymeric substances (EPSs). The aim of this study was to investigate the influence of forces of staphylococcal adhesion to different biomaterials on icaA (which regulates the production of EPS matrix components) and cidA (which is associated with cell lysis and extracellular DNA [eDNA] release) gene expression in Staphylococcus aureus biofilms. Experiments were performed with S. aureus ATCC 12600 and its isogenic mutant, S. aureus ATCC 12600 Δpbp4, deficient in peptidoglycan cross-linking. Deletion of pbp4 was associated with greater cell wall deformability, while it did not affect the planktonic growth rate, biofilm formation, cell surface hydrophobicity, or zeta potential of the strains. The adhesion forces of S. aureus ATCC 12600 were the strongest on polyethylene (4.9 ± 0.5 nN), intermediate on polymethylmethacrylate (3.1 ± 0.7 nN), and the weakest on stainless steel (1.3 ± 0.2 nN). The production of poly-N-acetylglucosamine, eDNA presence, and expression of icaA genes decreased with increasing adhesion forces. However, no relation between adhesion forces and cidA expression was observed. The adhesion forces of the isogenic mutant S. aureus ATCC 12600 Δpbp4 (deficient in peptidoglycan cross-linking) were much weaker than those of the parent strain and did not show any correlation with the production of poly-N-acetylglucosamine, eDNA presence, or expression of the icaA and cidA genes. This suggests that adhesion forces modulate the production of the matrix molecule poly-N-acetylglucosamine, eDNA presence, and icaA gene expression by inducing nanoscale cell wall deformation, with cross-linked peptidoglycan layers playing a pivotal role in this adhesion force sensing. PMID:25746995

  12. Influence of Adhesion Force on icaA and cidA Gene Expression and Production of Matrix Components in Staphylococcus aureus Biofilms

    PubMed Central

    Harapanahalli, Akshay K.; Chen, Yun; Li, Jiuyi; Busscher, Henk J.

    2015-01-01

    The majority of human infections are caused by biofilms. The biofilm mode of growth enhances the pathogenicity of Staphylococcus spp. considerably, because once they adhere, staphylococci embed themselves in a protective, self-produced matrix of extracellular polymeric substances (EPSs). The aim of this study was to investigate the influence of forces of staphylococcal adhesion to different biomaterials on icaA (which regulates the production of EPS matrix components) and cidA (which is associated with cell lysis and extracellular DNA [eDNA] release) gene expression in Staphylococcus aureus biofilms. Experiments were performed with S. aureus ATCC 12600 and its isogenic mutant, S. aureus ATCC 12600 Δpbp4, deficient in peptidoglycan cross-linking. Deletion of pbp4 was associated with greater cell wall deformability, while it did not affect the planktonic growth rate, biofilm formation, cell surface hydrophobicity, or zeta potential of the strains. The adhesion forces of S. aureus ATCC 12600 were the strongest on polyethylene (4.9 ± 0.5 nN), intermediate on polymethylmethacrylate (3.1 ± 0.7 nN), and the weakest on stainless steel (1.3 ± 0.2 nN). The production of poly-N-acetylglucosamine, eDNA presence, and expression of icaA genes decreased with increasing adhesion forces. However, no relation between adhesion forces and cidA expression was observed. The adhesion forces of the isogenic mutant S. aureus ATCC 12600 Δpbp4 (deficient in peptidoglycan cross-linking) were much weaker than those of the parent strain and did not show any correlation with the production of poly-N-acetylglucosamine, eDNA presence, or expression of the icaA and cidA genes. This suggests that adhesion forces modulate the production of the matrix molecule poly-N-acetylglucosamine, eDNA presence, and icaA gene expression by inducing nanoscale cell wall deformation, with cross-linked peptidoglycan layers playing a pivotal role in this adhesion force sensing. PMID:25746995

  13. Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

    PubMed Central

    de la Fuente-Núñez, César; Mansour, Sarah C.; Wang, Zhejun; Jiang, Lucy; Breidenstein, Elena B.M.; Elliott, Melissa; Reffuveille, Fany; Speert, David P.; Reckseidler-Zenteno, Shauna L.; Shen, Ya; Haapasalo, Markus; Hancock, Robert E.W.

    2014-01-01

    Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients. PMID:26221537

  14. A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model.

    PubMed

    Estellés, Angeles; Woischnig, Anne-Kathrin; Liu, Keyi; Stephenson, Robert; Lomongsod, Evelene; Nguyen, Da; Zhang, Jianzhong; Heidecker, Manfred; Yang, Yifan; Simon, Reyna J; Tenorio, Edgar; Ellsworth, Stote; Leighton, Anton; Ryser, Stefan; Gremmelmaier, Nina Khanna; Kauvar, Lawrence M

    2016-04-01

    Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observedin vitroat an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus aureus(MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections. PMID:26833157

  15. The ability of S.aureus to form biofilm on the Ti-6Al-7Nb scaffolds produced by Selective Laser Melting and subjected to the different types of surface modifications.

    PubMed

    Szymczyk, Patrycja; Junka, Adam; Ziółkowski, Grzegorz; Smutnicka, Danuta; Bartoszewicz, Marzenna; Chlebus, Edward

    2013-01-01

    The Gram-positive coccus, Staphylococcus aureus, is the leading etiologic agent of limb and life-threatening biofilm-related infections in the patients following the orthopaedic implantations. The aim of the present paper is to estimate the ability of S. aureus to form biofilm on titanium alloy (Ti-6Al-7Nb) scaffolds produced by Selective Laser Melting (SLM) and subjected to the different types of surface modifications, including ultrasonic cleaning and chemical polishing. The results obtained indicate significantly the decreased ability of S.aureus to form biofilm on the surface of scaffolds subjected to the chemical polishing in comparison to the scaffolds cleaned ultrasonically. The data provided can be useful for future applications of the SLM technology in production of Ti-6Al-7Nb medical implants. PMID:23957680

  16. Efficacy of ultraviolet C light at sublethal dose in combination with antistaphylococcal antibiotics to disinfect catheter biofilms of methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis in vitro

    PubMed Central

    El-Azizi, Mohamed; Khardori, Nancy

    2016-01-01

    Background Biofilm formation inside inserted medical devices leads to their failure and acts as a source of refractory infections. The ultraviolet C (UVC) light is a potential therapy that can be used against the biofilm of bacterial pathogens. Objective We evaluated the efficacy of sublethal dose of UVC light with anti-staphylococcal antibiotics against biofilms made from 30 isolates of methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus and S. epidermidis on vascular catheters. Materials and methods A novel biofilm device was used to assess the combined approach. The biofilms on the catheters were irradiated with the UVC light at 254 nm and irradiance of 6.4 mW followed by treatment with vancomycin or quinupristin/dalfopristin at twice their minimum bactericidal concentrations or with linezolid at 64 µg/mL for 24 hours. The catheters were cut into segments and sonicated, and the number of the sessile cells was determined colorimetrically using XTT viable cells assay. The effect of UVC radiation followed by treatment with an antistaphylococcal antibiotic on the viability of the bacteria in the biofilm was visualized using LIVE/DEAD BacLight bacterial viability stain and confocal laser scanning microscopy. Results Exposure of the bacterial biofilms to the UVC light or each of the antibiotics alone was ineffective in killing the bacteria. Treatment of the biofilms with the antibiotics following their exposure to UVC light significantly (P<0.001) reduced the number of viable cells within the biofilms but did not completely eradicate them. Conclusion To our knowledge, this combinatorial approach has not been investigated before. The combined approach can be used as a therapeutic modality for managing biofilm-associated infections by preventing the establishment of biofilms and/or disrupting the formed biofilms on the inserted medical devices with the goal of increasing their usefulness and preventing infectious complications. Further

  17. Identification of the amino acids essential for LytSR-mediated signal transduction in Staphylococcus aureus and their roles in biofilm-specific gene expression

    PubMed Central

    Lehman, McKenzie K.; Bose, Jeffrey L.; Sharma-Kuinkel, Batu K.; Moormeier, Derek E.; Endres, Jennifer L.; Sadykov, Marat R.; Biswas, Indranil; Bayles, Kenneth W.

    2015-01-01

    Summary Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically expressed within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, over activation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events. PMID:25491472

  18. Evaluation of Antimicrobial Effects of Different Concentrations of Triple Antibiotic Paste on Mature Biofilm of Enterococcus faecalis

    PubMed Central

    Frough Reyhani, Mohammad; Rahimi, Saeed; Fathi, Zahra; Shakouie, Sahar; Salem Milani, Amin; Soroush Barhaghi, Mohammad Hossein; Shokri, Javad

    2015-01-01

    Background and aims. Triple antibiotic paste (TAP) is widely used in endodontics for root canal disinfection, particularly in regenerative procedures. The aim of this in vitro study was to evaluate the antimicrobial effects of different concentrations of TAP at 1-, 2-, 3-, and 4-week intervals on mature Enterococcus faecalis biofilm. Materials and methods. A total of 287 extracted one-rooted human central incisors were infected with E. faecalis ATCC 29212 after removing the crown and preparation. The root canal space was filled with one of the 0.01-, 0.1-, 1-, 10-, 100-, and 1000-mg/mL concentrations of TAP or normal saline (control). The root canal dentin was sampled after 1, 2, 3, and 4 weeks. The dentinal shavings were cultured on Mueller-Hinton agar plates after serial dilutions. The classic colony-forming unit (CFU) counting technique was used to determine remaining bacterial counts. Data were analyzed by using the two-way ANOVA, post hoc Tukey tests and one-way ANOVA (P<0.05). Results. TAP completely eliminated E. faecalis biofilms at all the intervals at concentrations of 1000, 100, and 10 mg/mL, whereas 1-, 0.1-, and 0.01-mg/mL TAP resulted in significant reduction of CFU means compared with the control group. There were no statistically significant differences between the four time intervals. Conclusion. Use of lower concentrations of TAP at short term could eradicate E. faecalis biofilm and decrease high-concentration side effects. PMID:26697145

  19. Evaluation of Antimicrobial Effects of Different Concentrations of Triple Antibiotic Paste on Mature Biofilm of Enterococcus faecalis.

    PubMed

    Frough Reyhani, Mohammad; Rahimi, Saeed; Fathi, Zahra; Shakouie, Sahar; Salem Milani, Amin; Soroush Barhaghi, Mohammad Hossein; Shokri, Javad

    2015-01-01

    Background and aims. Triple antibiotic paste (TAP) is widely used in endodontics for root canal disinfection, particularly in regenerative procedures. The aim of this in vitro study was to evaluate the antimicrobial effects of different concentrations of TAP at 1-, 2-, 3-, and 4-week intervals on mature Enterococcus faecalis biofilm. Materials and methods. A total of 287 extracted one-rooted human central incisors were infected with E. faecalis ATCC 29212 after removing the crown and preparation. The root canal space was filled with one of the 0.01-, 0.1-, 1-, 10-, 100-, and 1000-mg/mL concentrations of TAP or normal saline (control). The root canal dentin was sampled after 1, 2, 3, and 4 weeks. The dentinal shavings were cultured on Mueller-Hinton agar plates after serial dilutions. The classic colony-forming unit (CFU) counting technique was used to determine remaining bacterial counts. Data were analyzed by using the two-way ANOVA, post hoc Tukey tests and one-way ANOVA (P<0.05). Results. TAP completely eliminated E. faecalis biofilms at all the intervals at concentrations of 1000, 100, and 10 mg/mL, whereas 1-, 0.1-, and 0.01-mg/mL TAP resulted in significant reduction of CFU means compared with the control group. There were no statistically significant differences between the four time intervals. Conclusion. Use of lower concentrations of TAP at short term could eradicate E. faecalis biofilm and decrease high-concentration side effects. PMID:26697145

  20. An in vivo safety and efficacy demonstration of a topical liposomal nitric oxide donor treatment for Staphylococcus aureus biofilm-associated rhinosinusitis.

    PubMed

    Jardeleza, Camille; Thierry, Benjamin; Rao, Shasha; Rajiv, Sukanya; Drilling, Amanda; Miljkovic, Dijana; Paramasivan, Sathish; James, Craig; Dong, Dong; Thomas, Nicky; Vreugde, Sarah; Prestidge, Clive A; Wormald, Peter-John

    2015-12-01

    The burden of drug resistance emerges in the wake of chronic and repeated antibiotic use. This underpins the importance of discovering alternatives to current antibiotic regimens. In chronic rhinosinusitis (CRS), topical therapy such as nasal douches and steroid sprays is the mainstay of treatment. However, bacterial sinusitis such as those with Staphylococcus aureus biofilm infection point to more recalcitrant CRS subtypes, focusing research efforts into topical antimicrobial therapies. In the sinuses, both local mucosal and systemic effects must be considered in designing any new topical medication. Nitric oxide (NO), an endogenous antimicrobial agent, is found at extremely low levels in CRS sinuses and high levels in healthy sinuses. As a novel treatment modality, we have designed a liposomal formulation of an NO donor (LFNO) using isosorbide mononitrate, as a topical sinus wash in a sheep model of S. aureus biofilm rhinosinusitis. Heart rate (HR), blood pressure, mean arterial pressure (MAP), and histologic and ciliary analyses were assessed in the safety component. Efficacy was assessed by quantifying biofilm biomass post-treatment. LFNO-treated sheep had lesser inflammation (P = 0.02), and comparable ciliary preservation (P = 0.86) than the control group. A transient increase in HR and decrease in MAP were observed in the LFNO group (P < 0.05), but this was not accompanied by observable side effects. LFNO sheep had significantly lower biofilm biomass vs controls (P = 0.044). Our findings demonstrate the localized and systemic safety of LFNO in an animal model despite using high NO concentrations, thus warranting further investigation for its possible therapeutic role in CRS. PMID:26166254

  1. Observed Antagonistic Effect of Linezolid on Daptomycin or Vancomycin Activity against Biofilm-Forming Methicillin-Resistant Staphylococcus aureus in an In Vitro Pharmacodynamic Model

    PubMed Central

    Luther, Megan K.

    2015-01-01

    Pharmacodynamic activity in antibiotic combinations of daptomycin, vancomycin, and linezolid was investigated in a 48-h in vitro pharmacodynamic model. Using human-simulated free drug concentrations, activity against clinical biofilm-forming methicillin-resistant Staphylococcus aureus isolates was evaluated. Linezolid antagonized vancomycin activity at 24 and 48 h. Linezolid antagonized daptomycin at 24 and 48 h depending on dose and strain. Adding daptomycin increased vancomycin activity at 48 h (P < 0.03). These results may be strain dependent and require further clinical investigation. PMID:26369963

  2. Herpes Simplex Virus (HSV) Modulation of Staphylococcus aureus and Candida albicans Initiation of HeLa 299 Cell-Associated Biofilm.

    PubMed

    Plotkin, Balbina J; Sigar, Ira M; Tiwari, Vaibhav; Halkyard, Scott

    2016-05-01

    Although herpes simplex virus type-1 (HSV-1), and type-2 (HSV-2), Staphylococcus aureus and Candida albicans co-habit the oral and genital mucosa, their interaction is poorly understood. We determined the effect HSV has on bacterial and/or fungal adherence, the initial step in biofilm formation. HeLa229 cells were infected with HSV-1 (KOS) gL86 or HSV-2 (KOS) 333gJ (-) at a multiplicity of infection (MOI) of 50 and 10. S. aureus (ATCC 25923) and/or C. albicans (yeast forms or germ tube forms) were co-incubated for 30 min (37 °C; 5 % CO2; 5:1 organism: HeLa cell ratio; n = 16) with virus-infected HeLa cells or uninfected HeLa cell controls. Post-incubation, the monolayers were washed (3x; PBS), lysed (RIPA), and the lysate plated onto Fungisel and/or mannitol salts agar for standard colony count. The level of HeLa-associated S. aureus was significantly decreased (P < 0.05) for both HSV-1- and HSV-2-infected cells, as compared to virus-free HeLa cell controls (38 and 59 % of control, respectively). In contrast, HSV-1 and HSV-2 significantly (P < 0.05) enhanced HeLa cell association of C. albicans yeast forms and germ tube approximately two-fold, respectively. The effect of S. aureus on germ tube and yeast form adherence to HSV-1- and HSV-2-infected cells was specific for the Candida phenotype tested. Our study suggests that HSV, while antagonist towards S. aureus adherence enhances Candida adherence. Furthermore, the combination of the three pathogens results in S. aureus adherence that is either unaffected, or partially restored depending on both the herpes viral species and the fungal phenotype present. PMID:26758707

  3. Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

    PubMed

    Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

    2016-08-01

    The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. PMID:27238459

  4. Host Physiologic Changes Induced by Influenza A Virus Lead to Staphylococcus aureus Biofilm Dispersion and Transition from Asymptomatic Colonization to Invasive Disease

    PubMed Central

    Reddinger, Ryan M.; Luke-Marshall, Nicole R.

    2016-01-01

    ABSTRACT Staphylococcus aureus is a ubiquitous opportunistic human pathogen and a major health concern worldwide, causing a wide variety of diseases from mild skin infections to systemic disease. S. aureus is a major source of severe secondary bacterial pneumonia after influenza A virus infection, which causes widespread morbidity and mortality. While the phenomenon of secondary bacterial pneumonia is well established, the mechanisms behind the transition from asymptomatic colonization to invasive staphylococcal disease following viral infection remains unknown. In this report, we have shown that S. aureus biofilms, grown on an upper respiratory epithelial substratum, disperse in response to host physiologic changes related to viral infection, such as febrile range temperatures, exogenous ATP, norepinephrine, and increased glucose. Mice that were colonized with S. aureus and subsequently exposed to these physiologic stimuli or influenza A virus coinfection developed pronounced pneumonia. This study provides novel insight into the transition from colonization to invasive disease, providing a better understanding of the events involved in the pathogenesis of secondary staphylococcal pneumonia. PMID:27507829

  5. Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

    PubMed

    Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

    2016-05-01

    β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action. PMID:27015018

  6. Antimicrobial Resistance, Biofilm Formation and mecA Characterization of Methicillin-Susceptible S. aureus and Non-S. aureus of Beef Meat Origin in Egypt.

    PubMed

    Osman, Kamelia M; Amer, Aziza M; Badr, Jihan M; Helmy, Nashwa M; Elhelw, Rehab A; Orabi, Ahmed; Bakry, Magdy; Saad, Aalaa S A

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) have been found in various farm animal species throughout the world. Yet, methicillin-susceptible S. aureus (MSSA), methicillin-susceptible non-S. aureus (MS-NSA), and methicillin-resistant non-S. aureus (MR-NSA) were not investigated. Therefore, we persued to determine the diversity in their phenotypic virulence assay, phenotypic antimicrobial resistance profile and molecular characterization in one of the food chains in Egypt. Samples were collected during 2013 from beef meat at retail. Twenty seven isolates comprising five species (S. hyicus, S. aureus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus) were characterized for their antibiotic resistance phenotypic profile and antibiotic resistance genes (mecA, cfr, gyrA, gyrB, and grlA). Out of the 27 Staphylococcus isolates only one isolate was resistant to the 12 antibiotics representing nine classes. Raw beef meat sold across the Great Cairo zone, contains 66.7% of MRS, with highest prevalence was reported in S. aureus (66.7%), while the MRS non-S. aureus strains constituted 66.7% from which S. hyicus (60%), S. intermedius (33.3%), S. schleiferi subsp. coagulans (100%), and S. lentus (100%) were MRS. Seven S. aureus, six S. hyicus, four S. schleiferi subsp. coagulans, three S. intermedius, and one S. lentus isolates although being resistant to oxacillin yet, 11/27 (40.7%) carried the mecA gene. At the same time, the cfr gene was present in 2 of the nine S. aureus isolates, and totally undetectable in S. hyicus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus. Although, global researches largely focused into MRSA and MR-NSA in animals on pigs, the analysis of our results stipulates, that buffaloes and cattle could be MRSA dispersers and that this theme is not specific to pigs. Detection of MSSA virulence determinants is a must, as although oxacillin resistance may be absent yet, the MSSA may carry the virulence determinants which

  7. Antimicrobial Resistance, Biofilm Formation and mecA Characterization of Methicillin-Susceptible S. aureus and Non-S. aureus of Beef Meat Origin in Egypt

    PubMed Central

    Osman, Kamelia M.; Amer, Aziza M.; Badr, Jihan M.; Helmy, Nashwa M.; Elhelw, Rehab A.; Orabi, Ahmed; Bakry, Magdy; Saad, Aalaa S. A.

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) have been found in various farm animal species throughout the world. Yet, methicillin-susceptible S. aureus (MSSA), methicillin-susceptible non-S. aureus (MS-NSA), and methicillin-resistant non-S. aureus (MR-NSA) were not investigated. Therefore, we persued to determine the diversity in their phenotypic virulence assay, phenotypic antimicrobial resistance profile and molecular characterization in one of the food chains in Egypt. Samples were collected during 2013 from beef meat at retail. Twenty seven isolates comprising five species (S. hyicus, S. aureus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus) were characterized for their antibiotic resistance phenotypic profile and antibiotic resistance genes (mecA, cfr, gyrA, gyrB, and grlA). Out of the 27 Staphylococcus isolates only one isolate was resistant to the 12 antibiotics representing nine classes. Raw beef meat sold across the Great Cairo zone, contains 66.7% of MRS, with highest prevalence was reported in S. aureus (66.7%), while the MRS non-S. aureus strains constituted 66.7% from which S. hyicus (60%), S. intermedius (33.3%), S. schleiferi subsp. coagulans (100%), and S. lentus (100%) were MRS. Seven S. aureus, six S. hyicus, four S. schleiferi subsp. coagulans, three S. intermedius, and one S. lentus isolates although being resistant to oxacillin yet, 11/27 (40.7%) carried the mecA gene. At the same time, the cfr gene was present in 2 of the nine S. aureus isolates, and totally undetectable in S. hyicus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus. Although, global researches largely focused into MRSA and MR-NSA in animals on pigs, the analysis of our results stipulates, that buffaloes and cattle could be MRSA dispersers and that this theme is not specific to pigs. Detection of MSSA virulence determinants is a must, as although oxacillin resistance may be absent yet, the MSSA may carry the virulence determinants which

  8. Arginine Deiminase in Staphylococcus epidermidis Functions To Augment Biofilm Maturation through pH Homeostasis

    PubMed Central

    Lindgren, J. K.; Thomas, V. C.; Olson, M. E.; Chaudhari, S. S.; Nuxoll, A. S.; Schaeffer, C. R.; Lindgren, K. E.; Jones, J.; Zimmerman, M. C.; Dunman, P. M.; Bayles, K. W.

    2014-01-01

    Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids. PMID:24727224

  9. Biofilm-formation by Staphylococcus aureus and Staphylococcus epidermidis isolates from subclinical mastitis in conditions mimicking the udder environment.

    PubMed

    Seixas, R; Varanda, D; Bexiga, R; Tavares, L; Oliveira, M

    2015-01-01

    Staphylococcus is the genus most commonly isolated from bovine mastitis in many countries. It may express several virulence factors including biofilm formation, which may protect the bacterial community from antimicrobials' action, preventing these compounds from reaching its interior, where they reach subinhibitory concentrations (subMIC). Most biofilm production assays are performed in static conditions, while studies regarding antimicrobial resistance usually do not resemble the udder environment because they are performed at high concentrations. In this study we evaluated the influence of dynamic conditions and media, including Mueller Hinton Broth (MHB) and UHT whole milk (WM), as well as the effect of subMIC concentrations of five different antimicrobial agents on biofilm formation by staphylococci isolated from subclinical mastitis. Results suggest that dynamic conditions and media may influence biofilm formation and revealed that milking simulation may significantly increase biofilm production. Sub-MIC concentrations decrease biofilm formation in MHB but increase in WM, suggesting a protective role of milk against antimicrobial compounds' action. Therefore, in vitro conditions that simulate the udder environment and in vivo conditions should be included as one of the parameters in evaluation of biofilm producing strains, in order to provide more reliable results. PMID:26812821

  10. Efficacy of ciprofloxacin-clarithromycin combination against drug-resistant Pseudomonas aeruginosa mature biofilm using in vitro experimental model.

    PubMed

    Elkhatib, Walid; Noreddin, Ayman

    2014-12-01

    Pseudomonas aeruginosa is the main cause of mortality in cystic fibrosis patients and eradication of its biofilm represents a substantial problem clinically. In this study, biofilm of a cystic fibrosis strain P. aeruginosa PACI22 was established and confocal laser scanning microscopy was utilized for biofilm visualization. A quantitative time-kill biofilm model was implemented in vitro to assess the biocidal effect of ciprofloxacin, clarithromycin, and their combination at concentration levels ranged from 0.5× to 64× minimum biofilm inhibitory concentrations (MBIC) against the biofilm and the mean log bacterial densities (Log CFU/ml) retrieved from the biofilm were monitored by frequent sampling at 0, 3, 6, 9, 12, and 24 hr throughout the experiment. The results revealed that none of the tested antibiotics alone could completely eradicate the biofilm-ensconced bacteria at 0.5-64× MBIC values after 24 hr of treatment. Conversely, ciprofloxacin-clarithromycin combination at 32-64× MBIC entirely exterminated the biofilm. Furthermore, a substantial in vitro synergism between ciprofloxacin and clarithromycin against the biofilm was experimentally verified. This promising synergism affords scientific rationale for further in vivo investigations to evaluate the therapeutic potential of this combination for treatment of chronic pulmonary infections caused by P. aeruginosa biofilms. PMID:25050970